SDRD protein from Staphylococcus aureus and diagnostic kits including same

ABSTRACT

An isolated extracellular matrix-binding protein, designated as SdrD and its corresponding amino acid and nucleic acid sequences and motifs are described. The proteins, peptides, fragments thereof or antigenic portions thereof are useful for the prevention, inhibition, treatment and diagnosis of  S. aureus  infection and as scientific research tools. Further, antibodies or antibody fragments to the proteins, peptides, fragments thereof or antigenic portions thereof are also useful for the prevention, inhibition, treatment and diagnosis of  S. aureus  infection. In particular, the proteins or antibodies thereof may be administered to wounds or used to coat biomaterials to act as blocking agents to prevent or inhibit the binding of  S. aureus  to wounds or biomaterials.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 10/744,616, filed Dec. 24, 2003 now abandoned, which is adivisional application of U.S. application Ser. No. 09/200,650, filedNov. 25, 1998, now U.S. Pat. No. 6,680,195, and claims the benefit ofU.S. Provisional Application Nos. 60/066,815, filed Nov. 26, 1997 andSer. No. 60/098,427, filed Aug. 31, 1998, all of said applicationsincorporated herein by reference.

The U.S. Government has rights in this invention arising out of NationalInstitutes of Health grant number A120624.

FIELD OF THE INVENTION

The present invention is in the fields of microbiology and molecularbiology. The invention includes the isolation and use of extracellularmatrix-binding proteins and genes that express the proteins fromStaphylococcus aureus to inhibit, prevent and diagnose S. aureusinfection.

BACKGROUND OF THE INVENTION

In hospitalized patients Staphylococcus aureus is a major cause ofinfections associated with indwelling medical devices, such as cathetersand prostheses, and related infections of surgical wounds. A significantincrease in Staphylococcus aureus isolates that exhibit resistance tomost known antibiotics has been observed in hospitals throughout theworld. The recent emergence of resistance to vancomycin, the lastremaining antibiotic for treating methicillin-resistant Staphylococcusaureus (MRSA) infections, has emphasized the need for alternativeprophylactic or vaccine strategies to reduce the risk of nosocomial S.aureus infections.

Initial localized infections of wounds or indwelling medical devices canlead to serious invasive infections such as septicemia, osteomyelitis,and endocarditis. In infections associated with medical devices, plasticand metal surfaces become coated with host plasma and extracellularmatrix proteins such as fibrinogen and fibronectin shortly afterimplantation. The ability of S. aureus to adhere to these proteins is ofcrucial importance for initiating infection. Vascular grafts,intravenous catheters, artificial heart valves, and cardiac assistdevices are thrombogenic and prone to bacterial colonization. S. aureusis the most damaging pathogen to cause such infections.

Fibrin is the major component of blood clots, and fibrinogen/fibrin isone of the major plasma proteins deposited on implanted biomaterials.Considerable evidence exists to suggest that bacterial adherence tofibrinogen/fibrin is important in the initiation of device-relatedinfection. For example, as shown by Vaudaux et al., S. aureus adheres toin vitro plastic that has been coated with fibrinogen in adose-dependent manner (J. Infect. Dis. 160:865-875 (1989)). In addition,in a model that mimics a blood clot or damage to a heart valve, Herrmannet al. demonstrated that S. aureus binds avidly via a fibrinogen bridgeto platelets adhering to surfaces (J. Infect. Dis. 167: 312-322 (1993)).S. aureus can adhere directly to fibrinogen in blood clots formed invitro, and can adhere to cultured endothelial cells via fibrinogendeposited from plasma acting as a bridge (Moreillon et al., Infect.Immun. 63:4738-4743 (1995); Cheung et al., J. Clin. Invest. 87:2236-2245(1991)). As shown by Vaudaux et al. and Moreillon et al., mutantsdefective in the fibrinogen-binding protein clumping factor (ClfA)exhibit reduced adherence to fibrinogen in vitro, to explantedcatheters, to blood clots, and to damaged heart valves in the rat modelfor endocarditis (Vaudaux et al., Infect. Immun. 63:585-590 (1995);Moreillon et al., Infect. Immun. 63: 4738-4743 (1995)).

An adhesin for fibrinogen, often referred to as “clumping factor,” islocated on the surface of S. aureus cells. The interaction between theclumping factor on bacteria and fibrinogen in solution results in theinstantaneous clumping of bacterial cells. The binding site onfibrinogen is located in the C-terminus of the gamma chain of thedimeric fibrinogen glycoprotein. The affinity is very high and clumpingoccurs in low concentrations of fibrinogen. Scientists have recentlyshown that clumping factor also promotes adherence to solid phasefibrinogen, to blood clots, and to damaged heart valves (McDevitt etal., Mol. Microbiol. 11: 237-248 (1994); Vaudaux et al., Infect. Immun.63:585-590 (1995); Moreillon et al., Infect. Immun. 63: 4738-4743(1995)).

The gene for a clumping factor protein, designated ClfA, has beencloned, sequenced and analyzed in detail at the molecular level(McDevitt et al., Mol. Microbiol. 11: 237-248 (1994); McDevitt et al.,Mol. Microbiol. 16:895-907 (1995)). The predicted protein is composed of933 amino acids. A signal sequence of 39 residues occurs at theN-terminus followed by a 520 residue region (region A), which containsthe fibrinogen binding domain. A 308 residue region (region R), composedof 154 repeats of the dipeptide serine-aspartate, follows. The R regionsequence is encoded by the 18 basepair repeat GAYTCNGAYT CNGAYAGY (SEQID NO: 9) in which Y equals pyrimidines and N equals any base. TheC-terminus of ClfA has features present in many surface proteins ofGram-positive bacteria such as an LPDTG (SEQ ID NO: 10) motif, which isresponsible for anchoring the protein to the cell wall, a membraneanchor, and positive charged residues at the extreme C-terminus.

The platelet integrin alpha IIbβ3 recognizes the C-terminus of the gammachain of fibrinogen. This is a crucial event in the initiation of bloodclotting during coagulation. ClfA and alpha IIbβ3 appear to recognizeprecisely the same sites on fibrinogen gamma chain because ClfA canblock platelet aggregation, and a peptide corresponding to theC-terminus of the gamma chain (198-411) can block both the integrin andClfA interacting with fibrinogen (McDevitt et al., Eur. J. Biochem.247:416-424 (1997)). The fibrinogen binding site of alpha IIbβ3 is closeto, or overlaps, a Ca²⁺ binding determinant referred to as an “EF hand”.ClfA region A carries several EF hand-like motifs. A concentration ofCa²⁺ in the range of 3-5 mM blocks these ClfA-fibrinogen interactionsand changes the secondary structure of the ClfA protein. Mutationsaffecting the ClfA EF hand reduce or prevent interactions withfibrinogen. Ca²⁺ and the fibrinogen gamma chain seem to bind to thesame, or to overlapping, sites in ClfA region A.

The alpha chain of the leucocyte integrin, alpha Mβ2, has an insertionof 200 amino acids (A or I domain) which is responsible for ligandbinding activities. A novel metal ion-dependent adhesion site (MIDAS)motif in the I domain is required for ligand binding. Among the ligandsrecognized is fibrinogen. The binding site on fibrinogen is in the gammachain (residues 190-202). It was recently reported that Candida albicanshas a surface protein, alpha Intlp, having properties reminiscent ofeukaryotic integrins. The surface protein has amino acid sequencehomology with the I domain of alpha Mβ2, including the MIDAS motif.Furthermore, alpha Intlp binds to fibrinogen.

ClfA region A also exhibits some degree of sequence homology with alphaIntlp. Examination of the ClfA region A sequence has revealed apotential MIDAS motif Mutations in supposed cation coordinating residuesin the DXSXS (SEQ ID NO: 13) portion of the MIDAS motif in ClfA resultsin a significant reduction in fibrinogen binding. A peptidecorresponding to the gamma-chain binding site for alpha Mβ2 (190-202)has been shown by O′Connell et al. to inhibit ClfA-fibrinogeninteractions (O′Connell et al., J. Biol. Chem., in press). Thus itappears that ClfA can bind to the gamma-chain of fibrinogen at twoseparate sites. The ligand binding sites on ClfA are similar to thoseemployed by eukaryotic integrins and involve divalent cation bindingEF-hand and MIDAS motifs.

Scientists have recently shown that S. aureus expresses proteins otherthan ClfA that may bind fibrinogen (Boden and Flock, Mol. Microbiol.12:599-606 (1994)). One of these proteins is probably the same as thebroad spectrum ligand-binding protein reported by Homonylo-McGavin etal., Infect. Immun. 61:2479-2485 (1993). Another is coagulase, asreported by Boden and Flock, Infect. Immun. 57:2358-2363 (1989), apredominantly extracellular protein that activates the plasma clottingactivity of prothrombin. Coagulase binds prothrombin at its N-terminusand also interacts with soluble fibrinogen at its C-terminus Cheung etal., Infect. Immun. 63:1914-1920 (1995) have described a variant ofcoagulase that binds fibrinogen. There is some evidence that coagulasecan contribute, in a minor way, to the ability of S. aureus cells tobind fibrinogen. As shown by Wolz et al., Infect. Immun. 64:3142-3147(1996), in an agr regulatory mutant, where coagulase is expressed at ahigh level, coagulase appears to contribute to the binding of solublefibrinogen to bacterial cells. Also, as shown by Dickinson et al.,Infect. Immun. 63:3143-3150 (1995), coagulase contributes in a minor wayto the attachment of S. aureus to plasma-coated surfaces under flow.However, it is clear that clumping factor ClfA is the majorsurface-located fibrinogen-binding protein responsible for bacterialattachment to immobilized fibrinogen/fibrin.

The identification and isolation of additional S. aureus extracellularmatrix binding proteins would be useful for the development oftherapies, diagnosis, prevention strategies and research tools for S.aureus infection.

Accordingly it is an object of the present invention to provide 15isolated cell-wall associated extracellular matrix-binding proteins ofS. aureus and active fragments thereof.

It is a further object of the invention to provide methods forpreventing, diagnosing, treating or monitoring the progress of therapyfor bacterial infections caused by S. aureus.

It is a further object of the present invention to provide isolated S.aureus surface proteins that are related in amino acid sequence to ClfAand are able to promote adhesion to the extracellular matrix or hostcells.

It is another object of the present invention to generate antisera andantibodies to cell-wall associated extracellular matrix-binding proteinsof S. aureus, or active fragments thereof.

It is a further object of the present invention to provide S. aureusvaccines, including a DNA vaccine.

It is a further object of the present invention to provide improvedmaterials and methods for detecting and differentiating S. aureusorganisms in clinical and laboratory settings.

It is a further object of the invention to provide nucleic acid probesand primers specific for S. aureus.

It is a further object of the invention to provide isolatedextracellular matrix-binding proteins or peptides of S. aureus.

SUMMARY OF THE INVENTION

Isolated extracellular matrix-binding proteins, designated ClfB, SdrC,SdrD and SdrE, and their corresponding amino acid and nucleic acidsequences and motifs are described. The proteins, peptides, fragmentsthereof or antigenic portions thereof are useful for the prevention,inhibition, treatment and diagnosis of S. aureus infection and asscientific research tools. Further, antibodies or antibody fragments tothe proteins, peptides, fragments thereof or antigenic portions thereofare also useful for the prevention, inhibition, treatment and diagnosisof S. aureus infection. The proteins, peptides, peptide fragments,antibodies, or antibody fragments can be administered in an effectiveamount to a patient in need thereof in any appropriate manner,preferably intravenously or otherwise by injection, to impart active orpassive immunity. In an alternative embodiment, the proteins orantibodies thereof can be administered to wounds or used to coatbiomaterials to act as blocking agents to prevent or inhibit the bindingof S. aureus to wounds or biomaterials.

Specifically, extracellular matrix-binding proteins from S. aureusdesignated as ClfB, SdrC, SdrD, and SdrE are provided.

ClfB is a fibrinogen binding protein. The nucleic acid and amino acidsequences of ClfB are provided in FIG. 5. The amino acid sequence ofClfB is SEQ ID NO: 1, and the nucleic acid sequence of ClfB is SEQ IDNO:2.

SdrC has been discovered to bind to several extracellular matrixproteins of the host, including for example, bone sialoprotein (BSP),decorin, plasmin, fibrinogen and vitronectin. The amino acid and nucleicacid sequences of SdrC are SEQ ID NOS:3 and 4 respectively and areprovided in FIG. 7.

Another of the discovered proteins, SdrD, binds at least vitronectin.The amino acid and nucleic acid sequences of SdrD are SEQ ID NOS:5 and 6respectively and are provided in FIG. 8.

SdrE binds to extracellular matrix proteins, for example, bonesialoprotein (BSP). The amino acid and nucleic acid sequences of SdrEare SEQ ID NOS:7 and 8 respectively and are provided in FIG. 9.

ClfB has a predicted molecular weight of approximately 88 kDa and anapparent molecular weight of approximately 124 kDa. ClfB is a cell-wallassociated protein and binds both soluble and immobilized fibrinogen. Inaddition, ClfB binds both the alpha and beta chains of fibrinogen andacts as a clumping factor. SdrC, SdrD and SdrE are cell-wall associatedproteins that exhibit cation-dependent ligand binding of extracellularmatrix proteins such as decorin, plasmin, fibrinogen, vitronectin andBSP.

It has been discovered that in the A region of SdrC, SdrD, SdrE, ClfA,and ClfB, there is highly conserved amino acid sequence that can be usedto derive a consensus TYTFTDYVD (SEQ ID NO: 18) motif (see FIG. 20). Themotif can be used in vaccines to impart broad spectrum immunity againstbacterial infections. The motif can also be used as an antigen in theproduction of monoclonal or polyclonal antibodies to impart broadspectrum passive immunity. In an alternative embodiment, any combinationof the variable sequence motif (T/I) (Y/F) (T/V) (F) (T) (D/N) (Y) (V)(D/N) can be used as an immunogen or antigen, or in the preparation ofantibodies.

The ClfB, SdrC, SdrD and SdrE proteins or the consensus or variablemotifs thereof are useful as scientific research tools to identify S.aureus binding sites on the extracellular matrix. They are furtheruseful as research tools to promote an understanding of the mechanismsof bacterial pathology and the development of antibacterial therapies.

The ClfB, SdrC, SdrD and SdrE nucleic acid sequences or selectedfragments thereof, including the sequences encoding the consensus orvariable motifs, are useful as nucleic acid probes for theidentification of other S. aureus extracellular matrix-binding proteins.Alternatively, the amino acid sequences of the proteins, or selectedfragments thereof, can be used as probes to identify the correspondingnucleic acid sequences.

The ClfB, SdrC, SdrD and SdrE nucleic acid sequences or the sequencesencoding the consensus or variable motifs are further useful aspolynucleotides which comprise contiguous nucleic acid sequences capableof being expressed. The nucleic acid sequences may be inserted into avector and placed in a microorganism for the production of recombinantClfB, SdrC, SdrD and SdrE proteins or the variable or consensus aminoacid motifs. This allows for the production of the gene product uponintroduction of said polynucleotide into eukaryotic tissues in vivo. Theencoded gene product preferably either acts as an immunostimulant or asan antigen capable of generating an immune response. Thus, the nucleicacid sequences in this embodiment encode an MSCRAMM (Microbial SurfaceComponents Recognising Adhesive Matrix Molecules) immunogenic epitope,and optionally a cytokine or a T-cell costimulatory element, such as amember of the B7 family of proteins.

There are several advantages of immunization with a gene rather than itsgene product. The first is the relative simplicity with which native ornearly native antigen can be presented to the immune system. A secondadvantage of DNA immunization is the potential for the immunogen toenter the MHC class I pathway and evoke a cytotoxic T cell response.Cell-mediated immunity is important in controlling infection. Since DNAimmunization can evoke both humoral and cell-mediated immune responses,its greatest advantage may be that it provides a relatively simplemethod to survey a large number of S. aureus genes for their vaccinepotential.

Antibodies immunoreactive with ClfB, SdrC, SdrD and SdrE proteins, ortheir active fragments, including with the consensus or variable aminoacid motifs, are provided herein. Vaccines or other pharmaceuticalcompositions containing the proteins or amino acid motifs areadditionally provided herein.

Antibodies and antisera to the consensus TYTFTDYVD (SEQ ID NO: 18)sequence epitope or the variable (T/I) (Y/F) (T/V) (F) (T) (D/N) (Y) (V)(D/N) sequence, specifically TYTFTNYVD (SEQ ID NO: 19) in SdrC,TYTFTDYVD (SEQ ID NO: 18) in SdrD and SdrE, TFVFTDYVN (SEQ ID NO: 20) inClfB or IYTFTDYVN (SEQ ID NO: 21) in ClfA are provided herein. Vaccinesor other pharmaceutical compositions containing the epitopes are alsoprovided herein.

In addition, diagnostic kits containing nucleic acid molecules, theproteins, antibodies or antisera to ClfB, SdrC, SdrD, SdrE or theiractive fragments, including the consensus or variable amino acid motifsand the appropriate reagents for reaction with a sample are alsoprovided.

In one embodiment of the invention, the diagnostic kit is used toidentify patients or animals that have levels of antibodies to ClfBClfB, SdrC, SdrD, or SdrE that are above a population norm. The plasmaof the patients or animals can be obtained, processed, and administeredto a host in need of passive immunity to S. aureus infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation comparing features of unprocessedClfA and ClfB proteins. S indicates the signal sequence. A indicates theconserved region (region A). P indicates the proline-rich region(repeats are indicated by gray boxes). R indicates the SD repeat region(region R). W indicates the wall-spanning region. M indicates themembrane spanning and anchoring regions. EF hand I of ClfA and itspartial homologue on ClfB are indicated by black vertical bars. TheMIDAS motifs are indicated by hatched (DXSXS) (SEQ ID NO: 13) and narrowvertical lines (downstream T and D residues) connected by dashed lines.

FIG. 2 is a schematic representation of general plasmid and probeconstructions for sequencing clfB. A repeat-carrying EcoRI fragment wascloned from phage clone A1-1 into pGEM 7Z (f)+ to give pA1-1E (top), andsubsequently reduced by deletion of an XbaI fragment to give pA1-1EX,which contains the entire clfB gene. A SmaI fragment containing clfB and500 by of upstream DNA was cloned into pCU1 for overexpression andcomplementation work (pA1-1EA). The HpaI probe used to screen mutants,and the hybridizing BamHI fragment are also indicated.

FIG. 3 is a schematic representation showing construction of a cassettefor allele replacement. clfB was interrupted by blunt-end cloning the Tcdeterminant from pT181 into the HpaI site in the middle of the gene inpA1-1EX. pTS2 was then cloned into the SmaI site of the cassette toenable temperature sensitive propagation in S. aureus.

FIG. 4 is a schematic representation of a physical map of the sdrC sdrDsdrE locus in S. aureus strain Newman. The extents of the plasmid clonesare delineated. A6-2 is a LambdaGEM®-12 clone. pEJ1, pEJ2 and pEJ3 areA6-2 fragments subcloned in the pGEM 7Z (f)+(pEJ 1 and pEJ2) and thepBluescript KS+vector (pEJ3). pC1 is a HindIII fragment directly clonedfrom strain Newman in the pBluescript KS+vector. Arrows indicate thedirection of transcription of sdrC, sdrD and sdrE.

FIGS. 5A-5D show the nucleic acid sequence of ClfB and flanking DNA, andamino acid translation of the ORF. The likely start codon is doubleunderlined, and the principal regions indicated using the abbreviationsof FIG. 1. Two salient features of region A, the DYSNS (SEQ ID NO: 11)of the putative MIDAS motif, and the sequence FTDYVN (SEQ ID NO: 12),the longest region of identity with ClfB, are underlined. Vertical barsindicate the repeats in the proline-rich region. An inverted repeatspecifying a possible transcription termination signal is underlined.

FIG. 6 is an amino acid sequence alignment of part of region A of theClfB and ClfA proteins in the region of strongest similarity. EF hand Iof ClfA is underlined. Identical residues are denoted by an asterisk;conservative substitutions are denoted by a period. The DXSXS (SEQ IDNO: 13) portion of the MIDAS motif of ClfB is double underlined.

FIGS. 7A-7D show the nucleic acid sequence and amino acid translation ofthe sdrC gene. The consensus TYTFTDYVD (SEQ ID NO: 18) motif, expressedin SdrC as TYTFTNYVD (SEQ ID NO: 19), the EF hands in the B repeats, andthe LPXTG (SEQ ID NO: 14) motif are underlined. Major regions, such asthe signal sequence (S), region A (A), B repeats (B) region R(R), thewall-spanning domain (W), and the membrane-anchoring domain (M), areindicated.

FIGS. 8A-8E show the nucleic acid sequence and amino acid translation ofthe sdrD gene. The consensus TYTFTDYVD (SEQ ID NO: 18) motif, the EFhands in the B repeats, and the LPXTG (SEQ ID NO: 14) motif areunderlined. Major regions, such as the signal sequence (S), region A(A), B repeats (B) region R(R), the wall-spanning domain (W), and themembrane-anchoring domain (M), are indicated.

FIGS. 9A-9D show the nucleic acid sequence and amino acid translation ofthe sdrE gene. The consensus TYTFTDYVD (SEQ ID NO: 18) motif, the EFhands in the B repeats, and the LPXTG (SEQ ID NO: 14) motif areunderlined. Major regions, such as the signal sequence (S), region A(A), B repeats (B) region R(R), the wall-spanning domain (W), and themembrane-anchoring domain (M), are indicated.

FIG. 10 is a schematic diagram of the region R-containing proteins.Numerals over the proteins denote numbers of amino acids in the regions,numerals under the proteins denote the location on the amino acidsequence of the motifs counted from the beginning of the signal peptide.Abbreviations: S: Signal peptide; A: Region A; B: B repeat; R: Region R;W. M: Wall and membrane spanning regions.

FIG. 11 is a chart showing similarities between A regions ClfA, ClfB,SdrC, SdrD and SdrE. Each sequence was aligned in pairwise combinationsand the percent identical residues given.

FIGS. 12A-12B show Clustal™ multiple sequence alignments of areas ofsimilarity of the A and B regions of the region R containing genes ofstrain Newman. An asterisk denotes identity of amino acids, and a colonrepresents increasing similarity of polarity andhydrophobicity/hydrophilicity of side chains of amino acids. Alignments1-4 show areas from region A. Alignments 1, 3 and 4 show the commonmotifs. Alignment 2 shows homology in the vicinity of the ClfA EF-hand(underlined), with the consensus TYTFTDYVD (SEQ ID NO: 18) sequenceconserved in all five genes. Alignment 5 shows the B repeats of proteinsSdrC, SdrD and SdrE with possible EF hands underlined.

FIG. 13 is a time-course graph of ClfB expression in S. aureus Newmanversus time, monitored by Western blotting. Shake flask cultures weresampled at specific time intervals. A standard number of cells was usedto prepare lysates.

FIG. 14 is a graph of absorbance versus concentration of ClfA/ClfBcomparing the binding of increasing concentrations of biotinylatedrecombinant region A from ClfA and ClfB to fibrinogen coated plates.Binding to BSA-coated plates is shown as a control. The closed squaresymbol represents fibrinogen-ClfA; the closed circle symbol representsfibrinogen-ClfB; the open square symbol represents BSA-ClfA; the opencircle symbol represents BSA-ClfB.

FIG. 15 is a graph of cells bound versus fibrinogen concentrationshowing adherence of S. aureus Newman and mutants to fibrinogenimmobilized on ELISA plates. Increasing amounts of fibrinogen were usedto coat the plates, and a fixed concentration of cells from exponentialphase cultures were added. The square symbol represents wild-type; thediamond symbol represents clfA; the circle symbol represents clfB; thetriangle symbol represents clfAclfB; the x symbol representsclfAclfB,clfB⁺.

FIG. 16 is a graph of cells bound versus fibrinogen concentrationshowing adherence of S. aureus Newman and mutants to fibrinogenimmobilized on ELISA plates. Increasing amounts of fibrinogen were usedto coat the plates, and a fixed concentration of cells from stationaryphase cultures added. The square symbol represents wild-type; thediamond symbol represents clfA; the circle symbol represents clfB; thetriangle symbol represents clfAclfB; the x symbol representsclfAclfB,ClfB⁺.

FIG. 17 is a graph of cells bound versus IgG concentration showingeffects of preincubation with anti-ClfB IgG on adherence of S. aureusNewman and mutants to immobilized fibrinogen. The square symbolrepresents wild-type; the diamond symbol represents clfA; the circlesymbol represents clfB; the x symbol represents clfAclfB,clfB⁺.

FIG. 18 is a bar graph showing adherence of S. aureus Newman and mutantsto explanted hemodialysis tubing. Cells from two hour shake-flaskcultures were used. The graph provides the means and SEM of threeexperiments.

FIG. 19 is a bar graph showing adherence of S. aureus Newman and mutantsto fibrinogen immobilized on PMMA (polymethylmethacrylate) coverslips.Cells from two hour shake-flask cultures were used. The graph providesthe means and SEM of three experiments.

FIG. 20 is a table which shows the highly conserved amino acid sequencesin the A region of ClfA, ClfB, SdrC, SdrD and SdrE, which are used toprovide consensus and variable motifs.

FIG. 21 is a graph of absorbance versus concentration of anti-TYTFTDYVD(SEQ ID NO: 18) antibodies, demonstrating the binding of increasingconcentrations of the antibodies to ClfA, ClfB or BSA coated plates.BSA-coated plates are used as a control, and no significant binding isobserved. The closed square symbol represents antibody bound to ClfB;the open diamond symbol represents antibody bound to ClfA; the opencircle symbol represents BSA.

FIG. 22 is a Western Blot which illustrates the differing specificitiesof ClfA and ClfB in the binding of human fibrinogen. The Western Blotwas created by the separation of human fibrinogen, and later, theincubation of the nitrocellulose membrane with the A region of eitherbiotinylated ClfA or ClfB. Biotinylated ClfA region A binds the y chainof fibrinogen, as is seen in lane A2. Biotinylated ClfB region A bindsto both the a and 13 chains of fibrinogen, as seen in lane B2.

FIG. 23 is a bar graph showing adherence of recombinant SdrC region A(SdrCA) to ten different extracellular matrix proteins, BSA, BSP, twoforms of collagen, decorin, fibrinogen, fibronectin, laminin, plasminand vitronectin. The extracellular matrix proteins were immobilized onmicrotiter wells. Absorbance tests revealed reactivity of SdrCA withfibrinogen, BSP, decorin, plasmin and vitronectin.

DETAILED DESCRIPTION OF THE INVENTION

Isolated extracellular matrix-binding proteins, designated ClfB, SdrC,SdrD and SdrE, and their corresponding amino acid and nucleic acidsequences and motifs are described. The proteins, peptides, fragmentsthereof or antigenic portions thereof are useful for the prevention,inhibition, treatment and diagnosis of S. aureus infection and asscientific research tools. Further, antibody or antibody fragments tothe proteins, peptides, fragments thereof or antigenic portions thereofare also useful for the prevention, inhibition, treatment and diagnosisof S. aureus infection. In particular, the proteins or antibodies, oractive fragments thereof may be administered as vaccines to induceeither passive or cellular immunity.

ClfB Binds to at Least Fibrinogen.

SdrC has been discovered to bind to extracellular matrix proteins of thehost, including for example, BSP, decorin, plasmin, vitronectin andfibrinogen. SdrD binds to at least vitronectin. SdrE binds toextracellular matrix proteins, for example, bone sialoprotein (BSP).

The amino acid sequence of ClfB is SEQ ID NO: 1. The nucleic acidsequence encoding ClfB is SEQ ID NO:2. The nucleic acid and amino acidsequences of ClfB are also provided in FIG. 5. The amino acid andnucleic acid sequences of SdrC are SEQ ID NOS:3 and 4 respectively andare provided in FIG. 7. The amino acid and nucleic acid sequences ofSdrD are SEQ ID NOS:5 and 6 respectively and are provided in FIG. 8. Theamino acid and nucleic acid sequences of SdrE are SEQ ID NOS:7 and 8respectively and are provided in FIG. 9. The term “isolated” is definedherein as free from at least some of the components with which itnaturally occurs. In a preferred embodiment, an isolated component is atleast 90% pure, and more preferably 95%.

ClfB has a predicted molecular weight of approximately 88 kDa and anapparent molecular weight of approximately 124 kDa. ClfB is a cell-wallassociated protein and binds both soluble and immobilized fibrinogen. Inaddition, ClfB binds both the alpha and beta chains of fibrinogen andacts as a clumping factor. Despite the low level of identity betweenClfA and ClfB, both proteins bind fibrinogen (on different chains) by amechanism that is susceptible to inhibition by divalent cations, despitenot sharing obvious metal binding motifs. The ClfB protein has beendemonstrated to be a virulence factor in experimental endocarditis.

The SdrC, SdrD and SdrE proteins are related in primary sequence andstructural organization to the ClfA and ClfB proteins and are localizedon the cell surface. The SdrC, SdrD and SdrE proteins are cellwall-associated proteins, having a signal sequence at the N-terminus andan LPXTG (SEQ ID NO: 14) motif, hydrophobic domain and positivelycharged residues at the C-terminus. Each also has an SD repeatcontaining region R of sufficient length to allow, along with the Bmotifs, efficient expression of the ligand binding domain region A onthe cell surface. With the A region of the SdrC, SdrD and SdrE proteinslocated on the cell surface, the proteins can interact with proteins inplasma, the extracellular matrix or with molecules on the surface ofhost cells. The Sdr proteins share some limited amino acid sequencesimilarity with ClfA and ClfB. Like ClfA and ClfB, SdrC, SdrD and SdrEalso exhibit cation-dependent ligand binding of extracellular matrixproteins.

It was surprising to learn that the disclosed extracellularmatrix-binding proteins share a unique dipeptide repeat region (regionR) including predominately aspartate and serine residues. It had beenreported by McDevitt et al., Mol. Microbiol. 11: 237-248 (1994);McDevitt et al., Mol. Microbiol. 16:895-907 (1995) that ClfA also hasthis R repeat region. He reported that that there were genes in S.epidermidus that hybridized to the gene encoding the R domain containingprotein. However, McDevitt et al did not know the function of the Rregion and had not discovered that other cell surface proteins from S.aureus, S. hemolyticus, S. lugdenensis, S. schleriferi share thisunusual motif. Therefore, in one aspect of this invention, a method isprovided for the identification of genes and encoding proteins from S.aureus (other than ClfA), S. hemolyticus, S. lugdenensis, S. schleriferiuseful for the prevention, treatment, and diagnosis of bacterialinfection that includes using the R repeat region as an identifyingprobe.

The DS repeat is encoded by 18 nucleotide repeats with the consensus(where Y equals pyrimidines and N equals any base) GAYTCNGAYT CNGAYAGY(SEQ ID NO: 9, with TCN as the first and second serine codons and AGY asthe third serine codon. The R region is near the C-terminus of theproteins and typically contains between 40 and 300 DS residues, or moreparticularly, greater than 40, 60, 80, 100, 125, 150, 200 or 250repeating units, of which greater than 90, 95 or even 98% of the aminoacids are D or S. The R region DS repeat varies in length betweenproteins, and while the R region itself does not bind extracellularmatrix proteins, the R region enables the presentation of the bindingregions of the protein on the cell surface of S. aureus. Thus, probes tothe consensus DNA encoding the DS repeat (see above) can be used toidentify other genes encoding different binding proteins essential tothe attachment of S. aureus to host tissues. Antibodies to an R regioncan be used to discover such additional binding proteins as well.

The sdr genes are closely linked and tandemly arrayed. The Sdr proteinshave both organizational and sequence similarity to ClfA and ClfB. Atthe N-terminus secretory signal sequences precede A regions which areapproximately 500 residues in length. The A regions of the Sdr and Clfproteins exhibit only 20-30% residue identity when aligned with anyother member of the family.

It has been discovered that in the A region of SdrC, SdrD, SdrE, ClfA,and ClfB, there is highly conserved amino acid sequence that can be usedto derive a consensus TYTFTDYVD (SEQ ID NO: 18) motif The motif exhibitsslight variation between the different proteins. This variation, alongwith the consensus sequence of the motif is depicted in FIG. 20. In theClf-Sdr proteins, this motif is highly conserved. The motif can be usedin vaccines to impart broad spectrum cellular immunity to bacterialinfections, and also can be used as an antigen in the production ofmonoclonal or polyclonal antibodies. Such an antibody can be used toimpart broad spectrum passive immunity.

The Sdr proteins differ from ClfA and ClfB by having two to fiveadditional 110-113 residue repeated sequences (B-motifs) located betweenregion A and the R-region. Each B-motif contains a consensusCa²⁺-binding EF-hand loop normally found in eukaryotic proteins. Thestructural integrity of a recombinant protein comprising the fiveB-repeats of SdrD was shown by bisANS fluorescence analysis to beCa²⁺-dependent, suggesting that the EF-hands are functional. When Ca²⁺was removed the structure collapsed to an unfolded conformation. Theoriginal structure was restored by addition of Ca²⁺. The C-terminalR-domains of the Sdr proteins contain 132-170 SD residues. These arefollowed by conserved wall-anchoring regions characteristic of manysurface proteins of Gram positive bacteria. The sdr locus was present inall 31 S. aureus strains from human and bovine sources tested bySouthern hybridization, although in a few strains it contained tworather than three genes.

In the Sdr and Clf proteins this B motif is highly conserved while adegenerate version occurs in fibronectin binding MSCRAMMS, as well asthe collagen binding protein Cna. The B motifs, in conjunction with theR regions, are necessary for displaying the ligand-binding domain atsome distance from the cell surface.

The repeated B motifs are one common denominator of the sub-group of SDrepeat proteins described herein. These motifs are found in differentnumbers in the three Sdr proteins from strain Newman. There are cleardistinctions between the individual B motifs. The most conserved unitsare those located adjacent to the R regions (SdrC B2, SdrD B5 and SdrEB3). They differ from the rest at several sites, especially in theC-terminal half. A noteworthy structural detail is that adjacent Brepeats are always separated by a proline residue present in theC-terminal region, but a proline never occurs between the last B repeatsand the R region. Instead this linker is characterized by a short acidicstretch. These differences are evidence that the end units have adifferent structural or functional role compared to the other B motifs.The N-terminal B motifs of SdrD and SdrE have drifted apart from theothers, and there are numerous amino acid alterations, including smallinsertions and deletions whereas the remaining internal B motifs aremore highly conserved. Note that each of the three Sdr proteins has atleast one B motif of each kind.

The C-terminal R-domains of the Sdr proteins contain 132-170 SDresidues. These are followed by conserved wall-anchoring regionscharacteristic of many surface proteins of Gram positive bacteria.

ClfB, SdrC, SdrD and SdrE subdomains are shown in FIG. 10 and, dependingon the protein, include subdomains A and B1-B5.

The terms ClfB protein, SdrC protein, SdrD protein and SdrE protein aredefined herein to include ClfB, SdrC, SdrD and SdrE subdomains, andactive or antigenic fragments of ClfB, SdrC, SdrD and SdrE proteins,such as consensus or variable sequence amino acid motifs. Activefragments of ClfB, SdrC, SdrD, SdrE and consensus or variable sequenceamino acid motifs peptides or proteins are defined herein as peptides orpolypeptides capable of blocking the binding of S. aureus toextracellular matrix proteins. Antigenic fragments of ClfB, SdrC, SdrD,SdrE proteins or the consensus or variable amino acid motifs are definedherein as peptides or polypeptides capable of producing an immunologicalresponse.

Nucleic Acid Sequences

The nucleic acid sequences encoding ClfB, SdrC, SdrD, SdrE and theconsensus or variable sequence amino acid motifs are useful for theproduction of recombinant extracellular matrix-binding proteins. Theyare further useful as nucleic acid probes for the detection of S.aureus-binding proteins in a sample or specimen with high sensitivityand specificity. The probes can be used to detect the presence of S.aureus in the sample, diagnose infection with the disease, quantify theamount of S. aureus in the sample, or monitor the progress of therapiesused to treat the infection. The nucleic acid and amino acid sequencesare also useful as laboratory research tools to study the organism andthe disease, thus furthering the development of therapies and treatmentsfor the disease.

It will be understood by those skilled in the art that ClfB, SdrC, SdrD,SdrE and the consensus or variable sequence amino acid motifs are alsoencoded by sequences substantially similar to the nucleic acid sequencesprovided in the sequence listing. By “substantially similar” is meant aDNA sequence which, by virtue of the degeneracy of the genetic code, isnot identical with that shown in any of SEQ ID NOS: 2, 4, 6, and 8, butwhich still encodes the same amino acid sequence; or a DNA sequencewhich encodes a different amino acid sequence but retains the activitiesof the proteins, either because one amino acid is replaced with anothersimilar amino acid, or because the change (whether it be substitution,deletion or insertion) does not affect the active site of the protein.In the latter case, the sequence has substantial homology to thedisclosed sequence if it encodes a protein with at least 70% 80%, 90%,95% or even 98% of the same amino acids.

Also provided herein are sequences of nucleic acid molecules thatselectively hybridize with nucleic acid molecules encoding theextracellular matrix-binding proteins from S. aureus described herein orcomplementary sequences thereof. By “selective” or “selectively” ismeant a sequence which does not hybridize with other nucleic acids toprevent adequate detection of ClfB, SdrC, SdrD, SdrE or the consensus orvariable sequence amino acid motifs. Therefore, in the design ofhybridizing nucleic acids, selectivity will depend upon the othercomponents present in a sample. The hybridizing nucleic acid should haveat least 70% complementarity with the segment of the nucleic acid towhich it hybridizes. As used herein to describe nucleic acids, the term“selectively hybridizes” excludes the occasional randomly hybridizingnucleic acids, and thus, has the same meaning as “specificallyhybridizing”. The selectively hybridizing nucleic acids of the inventioncan have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, and 99%complementarity with the segment of the sequence to which it hybridizes.

The invention contemplates sequences, probes and primers whichselectively hybridize to the encoding DNA or the complementary, oropposite, strand of DNA as those specifically provided herein. Specifichybridization with nucleic acid can occur with minor modifications orsubstitutions in the nucleic acid, so long as functionalspecies-specific hybridization capability is maintained. By “probe” ismeant nucleic acid sequences that can be used as probes or primers forselective hybridization with complementary nucleic acid sequences fortheir detection or amplification, which probes can vary in length fromabout 5 to 100 nucleotides, or preferably from about 10 to 50nucleotides, or most preferably about 18-24 nucleotides. Therefore, theterms “probe” or “probes” as used herein are defined to include“primers”. Isolated nucleic acids are provided herein that selectivelyhybridize with the species-specific nucleic acids under stringentconditions and should have at least 5 nucleotides complementary to thesequence of interest as described by Sambrook, J., E. F. Fritsch, and T.Maniatis. 1989. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

If used as primers, the composition preferably includes at least twonucleic acid molecules which hybridize to different regions of thetarget molecule so as to amplify a desired region. Depending on thelength of the probe or primer, the target region can range between 70%complementary bases and full complementary and still hybridize understringent conditions. For example, for the purpose of diagnosing thepresence of the S. aureus, the degree of complementary between thehybridizing nucleic acid (probe or primer) and the sequence to which ithybridizes (e.g., S. aureus DNA from a sample) is at least enough todistinguish hybridization with a nucleic acid from other bacteria.

The nucleic acid sequences encoding ClfB, SdrC, SdrD, SdrE activefragments thereof or consensus or variable sequence amino acid motifscan be inserted into a vector, such as a plasmid, and recombinantlyexpressed in a living organism to produce recombinant ClfB, SdrC, SdrDand SdrE proteins or fragments thereof, such as consensus or variablesequence amino acid motifs. For example, DNA molecules producingrecombinant ClfB, SdrC, and both SdrD and SdrE were deposited inplasmids pA1-1EX, pC1 and lambda phage A6-2, respectively, at the NCIMBunder the Accession Nos. 40903, 40902 and 40904, respectively on Oct.13, 1997.

Methods for the Detection and Identification of S. aureus

Methods of using the nucleic acids described herein to detect andidentify the presence of S. aureus are provided. The methods are usefulfor diagnosing S. aureus infections and disease such as upperrespiratory tract infections (such as otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratoryinfections (such as emphysema, lung abscess), cardiac (such as infectiveendocarditis), gastrointestinal (such as secretory diarrhea, splenicabscess, retroperitoneal abscess), central nervous system (such ascerebral abscess), ocular (such as blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (such as epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (such asimpetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis, bone and joint (such as septic arthritis,osteomyelitis).

The method involves the steps of obtaining a sample suspected ofcontaining S. aureus The sample may be taken from an individual, such asa wound, blood, saliva, tissues, bone, muscle, cartilage, or skin. Thecells can then be lysed, and the DNA extracted, precipitated andamplified. Detection of S. aureus DNA is achieved by hybridizing theamplified DNA with a S. aureus probe that selectively hybridizes withthe DNA as described above. Detection of hybridization is indicative ofthe presence of S. aureus.

Preferably, detection of nucleic acid (e.g. probes or primers)hybridization can be facilitated by the use of detectable moieties. Forexample, the probes can be labeled with biotin and used in astreptavidin-coated microtiter plate assay. Other detectable moietiesinclude radioactive labeling, enzyme labeling, and fluorescent labeling,for example.

DNA may be detected directly or may be amplified enzymatically usingpolymerase chain reaction (PCR) or other amplification techniques priorto analysis. RNA or cDNA can be similarly detected. Increased ordecreased expression of ClfB, SdrC, SdrD, SdrE and consensus or variablesequence amino acid motifs can be measured using any of the methods wellknown in the art for the quantitation of nucleic acid molecules, suchas, amplification, PCR, RT-PCR, RNase protection, Northern blotting, andother hybridization methods.

Diagnostic assays which test for the presence of the ClfB or SdrC, SdrDor SdrE proteins, peptides, motifs, fragments thereof or antibodies toany of these may also be used to detect the presence of an infection.Assay techniques for determining protein or antibody levels in a sampleare well known to those skilled in the art and include methods such asradioimmunoasssay, Western blot analysis and ELISA (Enzyme-LinkedImmunosorbant Assay) assays.

Amino Acid Sequences

It will be understood by those skilled in the art that minor amino acidsubstitutions or deletions may be present in functional ClfB, SdrC,SdrD, SdrE and consensus or variable sequence amino acid motifs,peptides, proteins, or fragments thereof. The amino acid sequences setforth herein and substantially similar amino acid sequences can be usedto produce synthetic ClfB, SdrC, SdrD, SdrE and consensus or variablesequence amino acid motifs, peptides, proteins or active fragmentsthereof. Active ClfB, SdrC, SdrD, SdrE or consensus or variable sequenceamino acid motifs, peptide or protein fragments are defined herein asClfB, SdrC, SdrD, SdrE or consensus or variable sequence amino acidmotifs, portions or peptides that bind to extracellular matrix proteinsor compete with or prevent S. aureus organisms from binding toextracellular matrix proteins such as decorin, plasmin, fibrinogen,vitronectin or bone sialoprotein.

When used in conjunction with amino acid sequences, the term“substantially similar” means an amino acid sequence which is notidentical to SEQ ID NOS:1, 3, 5, or 7, but which produces a proteinhaving the same functionality and retaining the activities of ClfB,SdrC, SdrD, SdrE and consensus or variable sequence amino acid motifs,either because one amino acid is replaced with another similar aminoacid, or because the change (whether it be substitution, deletion orinsertion) does not affect the active site of the protein or peptide.Two amino acid sequences are “substantially homologous” when at leastabout 70%, (preferably at least about 80%, and most preferably at leastabout 90% or 95%) of the amino acids match over the defined length ofthe sequences.

Extracellular Matrix-Binding Protein Antibodies

The isolated, recombinant or synthetic ClfB, SdrC, SdrD, SdrE orconsensus or variable sequence amino acid motifs, or peptides or activefragments thereof or fusion proteins thereof, are useful as scientificresearch tools to identify S. aureus binding sites on the extracellularmatrix. This will promote an understanding of the mechanisms ofbacterial pathology and the development of antibacterial therapies.Furthermore, the isolated, recombinant or synthetic protein, orantigenic portions thereof (including epitope-bearing fragments), orfusion proteins thereof can be administered to humans or animals asimmunogens or antigens. It can be administered alone or in combinationwith an adjuvant, for the production of antisera reactive with ClfB,SdrC, SdrD, SdrE or motifs or peptides thereof. In addition, thepeptides or proteins can be used to screen antisera for hyperimmunepatients from whom can be derived antibodies having a very high affinityfor the proteins.

Antibodies isolated from the antisera are useful for the specificdetection of S. aureus or S. aureus extracellular matrix-bindingproteins or as research tools. The term “antibodies” as used hereinincludes monoclonal antibodies, polyclonal, chimeric, single chain,bispecific, simianized, and humanized antibodies as well as Fabfragments, including the products of an Fab immunoglobulin expressionlibrary.

Monoclonal antibodies are generated by methods well known to thoseskilled in the art. The preferred method is a modified version of themethod of Kearney, et al., J. Immunol. 123:1548-1558 (1979), which isincorporated by reference herein. Briefly, animals such as mice orrabbits are inoculated with the immunogen in adjuvant, and spleen cellsare harvested and mixed with a myeloma cell line, such as P3X63Ag8,653.The cells are induced to fuse by the addition of polyethylene glycol.Hybridomas are chemically selected by plating the cells in a selectionmedium containing hypoxanthine, aminopterin and thymidine (HAT).Hybridomas producing the preferred antibodies are cloned, expanded andstored frozen for future production.

Techniques for the production of single chain antibodies are known tothose skilled in the art and described in U.S. Pat. No. 4,946,778 andcan be used to produce single chain antibodies to the proteins describedherein. Phage display technology may be used to select antibody geneshaving binding activities for ClfB, SdrC, SdrD, SdrE, and consensus orvariable sequence amino acid motifs, or antigenic portions thereof, fromPCR-amplified v genes of lymphocytes from humans screened for havingantibodies to ClfB, SdrC, SdrD, SdrE or consensus or variable sequenceamino acid motifs or naive libraries. Bispecific antibodies have twoantigen binding domains wherein each domain is directed against adifferent epitope.

The antibody may be labeled directly with a detectable label foridentification and quantitation of S. aureus. Labels for use inimmunoassays are generally known to those skilled in the art and includeenzymes, radioisotopes, and fluorescent, luminescent and chromogenicsubstances including colored particles such as colloidal gold and latexbeads. Suitable immunoassays include enzyme-linked immunosorbent assays(ELISA).

Alternatively, the antibody may be labeled indirectly by reaction withlabeled substances that have an affinity for immunoglobulin, such asprotein A or G or second antibodies. The antibody may be conjugated witha second substance and detected with a labeled third substance having anaffinity for the second substance conjugated to the antibody. Forexample, the antibody may be conjugated to biotin and theantibody-biotin conjugate detected using labeled avidin or streptavidin.Similarly, the antibody may be conjugated to a hapten and theantibody-hapten conjugate detected using labeled anti-hapten antibody.These and other methods of labeling antibodies and assay conjugates arewell known to those skilled in the art.

Antibodies to the disclosed proteins may also be used in productionfacilities or laboratories to isolate additional quantities of theprotein, such as by affinity chromatography.

The proteins, or antigenic portions thereof, are useful in the diagnosisof S. aureus bacterial infections and in the development of anti-S.aureus vaccines for active or passive immunization. When administered toa wound or used to coat polymeric biomaterials in vitro and in vivo,both the proteins and antibodies thereof are useful as blocking agentsto prevent or inhibit the initial binding of S. aureus to the wound siteor biomaterials. Preferably, the antibody is modified so that it is lessimmunogenic in the patient to whom it is administered. For example, ifthe patient is a human, the antibody may be “humanized” by transplantingthe complimentarily determining regions of the hybridoma-derivedantibody into a human monoclonal antibody as described by Jones et al.,Nature 321:522-525 (1986) or Tempest et al. Biotechnology 9:266-273(1991).

Medical devices or polymeric biomaterials to be coated with theantibodies, proteins and active fragments described herein include, butare not limited to, staples, sutures, replacement heart valves, cardiacassist devices, hard and soft contact lenses, intraocular lens implants(anterior chamber, posterior chamber or phakic), other implants such ascorneal inlays, kerato-prostheses, vascular stents, epikeratophaliadevices, glaucoma shunts, retinal staples, scleral buckles, dentalprostheses, thyroplastic devices, laryngoplastic devices, vasculargrafts, soft and hard tissue prostheses including, but not limited to,pumps, electrical devices including stimulators and recorders, auditoryprostheses, pacemakers, artificial larynx, dental implants, mammaryimplants, penile implants, cranio/facial tendons, artificial joints,tendons, ligaments, menisci, and disks, artificial bones, artificialorgans including artificial pancreas, artificial hearts, artificiallimbs, and heart valves; stents, wires, guide wires, intravenous andcentral venous catheters, laser and balloon angioplasty devices,vascular and heart devices (tubes, catheters, balloons), ventricularassists, blood dialysis components, blood oxygenators,urethral/ureteral/urinary devices (Foley catheters, stents, tubes andballoons), airway catheters (endotracheal and tracheostomy tubes andcuffs), enteral feeding tubes (including nasogastric, intragastric andjejunal tubes), wound drainage tubes, tubes used to drain the bodycavities such as the pleural, peritoneal, cranial, and pericardialcavities, blood bags, test tubes, blood collection tubes, vacutainers,syringes, needles, pipettes, pipette tips, and blood tubing.

It will be understood by those skilled in the art that the term “coated”or “coating”, as used herein, means to apply the protein, antibody, oractive fragment to a surface of the device, preferably an outer surfacethat would be exposed to S. aureus infection. The surface of the deviceneed not be entirely covered by the protein, antibody or activefragment.

Immunological and Pharmaceutical Compositions

Immunological compositions, including vaccine, and other pharmaceuticalcompositions containing the ClfB, SdrC, SdrD, SdrE or consensus orvariable sequence amino acid motif, peptides or proteins are includedwithin the scope of the present invention. One or more of the ClfB,SdrC, SdrD, SdrE or consensus or variable sequence amino acid motif,peptides, proteins, or active or antigenic fragments thereof, or fusionproteins thereof can be formulated and packaged, alone or in combinationwith other antigens, using methods and materials known to those skilledin the art for vaccines. The immunological response may be usedtherapeutically or prophylactically and may provide antibody immunity orcellular immunity such as that produced by T lymphocytes such ascytotoxic T lymphocytes or CD4⁺T lymphocytes.

The immunological compositions, such as vaccines, and otherpharmaceutical compositions can be used alone or in combination withother blocking agents to protect against human and animal infectionscaused by S. aureus. In particular, the compositions can be used toprotect humans against endocarditis or to protect humans or ruminantsagainst mastitis caused by S. aureus infections. The vaccine can also beused to protect canine and equine animals against similar S. aureusinfections.

To enhance immunogenicity, the proteins may be conjugated to a carriermolecule. Suitable immunogenic carriers include proteins, polypeptidesor peptides such as albumin, hemocyanin, thyroglobulin and derivativesthereof, particularly bovine serum albumin (BSA) and keyhole limpethemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solidphases. Other protein derived or non-protein derived substances areknown to those skilled in the art. An immunogenic carrier typically hasa molecular weight of at least 1,000 daltons, preferably greater than10,000 daltons. Carrier molecules often contain a reactive group tofacilitate covalent conjugation to the hapten. The carboxylic acid groupor amine group of amino acids or the sugar groups of glycoproteins areoften used in this manner. Carriers lacking such groups can often bereacted with an appropriate chemical to produce them. Preferably, animmune response is produced when the immunogen is injected into animalssuch as mice, rabbits, rats, goats, sheep, guinea pigs, chickens, andother animals, most preferably mice and rabbits. Alternatively, amultiple antigenic peptide comprising multiple copies of the protein orpolypeptide, or an antigenically or immunologically equivalentpolypeptide may be sufficiently antigenic to improve immunogenicitywithout the use of a carrier.

The ClfB, SdrC, SdrD, SdrE or consensus or variable sequence amino acidmotif, peptide, protein or proteins may be administered with an adjuvantin an amount effective to enhance the immunogenic response against theconjugate. At this time, the only adjuvant widely used in humans hasbeen alum (aluminum phosphate or aluminum hydroxide). Saponin and itspurified component Quil A, Freund's complete adjuvant and otheradjuvants used in research and veterinary applications have toxicitieswhich limit their potential use in human vaccines. However, chemicallydefined preparations such as muramyl dipeptide, monophosphoryl lipid A,phospholipid conjugates such as those described by Goodman-Snitkoff etal. J. Immunol. 147:410-415 (1991) and incorporated by reference herein,encapsulation of the conjugate within a proteoliposome as described byMiller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated byreference herein, and encapsulation of the protein in lipid vesiclessuch as Novasome™ lipid vesicles (Micro Vescular Systems, Inc., Nashua,N.H.) may also be useful.

The term “vaccine” as used herein includes DNA vaccines in which thenucleic acid molecule encoding ClfB, SdrC, SdrD, SdrE and consensus orvariable sequence amino acid motifs, or nucleic acid molecules which arenot identical to the disclosed sequences, but which are substantiallyhomologous thereto and encode peptides or proteins which have the samefunctionality and activities, or antigenic portions thereof in apharmaceutical composition is administered to a patient. For geneticimmunization, suitable delivery methods known to those skilled in theart include direct injection of plasmid DNA into muscles (Wolff et al.,Hum. Mol. Genet. 1:363 (1992)), delivery of DNA complexed with specificprotein carriers (Wu et al., J. Biol. Chem. 264:16985 (1989),coprecipitation of DNA with calcium phosphate (Benvenisty and Reshef,Proc. Natl. Acad. Sci. 83:9551 (1986)), encapsulation of DNA inliposomes (Kaneda et al., Science 243:375 (1989)), particle bombardment(Tang et al., Nature 356:152 (1992) and Eisenbraun et al., DNA CellBiol. 12:791 (1993)), and in vivo infection using cloned retroviralvectors (Seeger et al., Proc. Natl. Acad. Sci. 81:5849, 1984).

Methods of Administration and Dose of Pharmaceutical Compositions

Pharmaceutical compositions containing the ClfB, SdrC, SdrD or SdrEproteins, nucleic acid molecules, antibodies, or fragments thereof maybe formulated in combination with a pharmaceutical carrier such assaline, dextrose, water, glycerol, ethanol, other therapeutic compounds,and combinations thereof. The formulation should be appropriate for themode of administration. The compositions are useful for interferingwith, modulating, or inhibiting S. aureus host cell binding interactionswith the extracellular matrix.

Suitable methods of administration include, but are not limited to,topical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal and intradermal administration.

For topical administration, the composition is formulated in the form ofan ointment, cream, gel, lotion, drops (such as eye drops and eardrops), or solution (such as mouthwash). Wound or surgical dressings,sutures and aerosols may be impregnated with the composition. Thecomposition may contain conventional additives, such as preservatives,solvents to promote penetration, and emollients. Topical formulationsmay also contain conventional carriers such as cream or ointment bases,ethanol, or oleyl alcohol.

In a preferred embodiment, a vaccine is packaged in a single dosage forimmunization by parenteral (i.e., intramuscular, intradermal orsubcutaneous) administration or nasopharyngeal (i.e., intranasal)administration. The vaccine is most preferably injected intramuscularlyinto the deltoid muscle. The vaccine is preferably combined with apharmaceutically acceptable carrier to facilitate administration. Thecarrier is usually water or a buffered saline, with or without apreservative. The vaccine may be lyophilized for resuspension at thetime of administration or in solution.

The carrier to which the protein may be conjugated may also be apolymeric delayed release system. Synthetic polymers are particularlyuseful in the formulation of a vaccine to effect the controlled releaseof antigens. For example, the polymerization of methyl methacrylate intospheres having diameters less than one micron has been reported byKreuter, J., MICROCAPSULES AND NANOPARTICLES IN MEDICINE ANDPHARMACOLOGY, M. Donbrow (Ed). CRC Press, p. 125-148.

Microencapsulation of the protein will also give a controlled release. Anumber of factors contribute to the selection of a particular polymerfor microencapsulation. The reproducibility of polymer synthesis and themicroencapsulation process, the cost of the microencapsulation materialsand process, the toxicological profile, the requirements for variablerelease kinetics and the physicochemical compatibility of the polymerand the antigens are all factors that must be considered. Examples ofuseful polymers are polycarbonates, polyesters, polyurethanes,polyorthoesters polyamides, poly(d,l-lactide-co-glycolide-) (PLGA) andother biodegradable polymers. The use of PLGA for the controlled releaseof antigen is reviewed by Eldridge, J. H., et al. CURRENT TOPICS INMICROBIOLOGY AND IMMUNOLOGY, 146:59-66 (1989).

One typical dose for human administration is from 0.01 mg/kg to 10mg/kg. Based on this range, equivalent dosages for heavier body weightscan be determined. The dose should be adjusted to suit the individual towhom the composition is administered and will vary with age, weight andmetabolism of the individual. The vaccine may additionally containstabilizers such as thimerosal (ethyl(2-mercaptobenzoate-S)mercurysodium salt) (Sigma Chemical Company, St. Louis, Mo.) or physiologicallyacceptable preservatives.

Protein-Label Conjugates

When labeled with a detectable biomolecule or chemical, theextracellular matrix-binding proteins described herein are useful forpurposes such as in vivo and in vitro diagnostics and laboratoryresearch. Various types of labels and methods of conjugating the labelsto the proteins are well known to those skilled in the art. Severalspecific labels are set forth below. The labels are particularly usefulwhen conjugated to a protein such as an antibody or receptor.

For example, the protein can be conjugated to a radiolabel such as, butnot restricted to, .³²P, ³H, .⁴C, ³⁵S, ¹²⁵I, or .¹³¹I. Detection of alabel can be by methods such as scintillation counting, gamma rayspectrometry or autoradiography.

Bioluminescent labels, such as derivatives of firefly luciferin, arealso useful. The bioluminescent substance is covalently bound to theprotein by conventional methods, and the labeled protein is detectedwhen an enzyme, such as luciferase, catalyzes a reaction with ATPcausing the bioluminescent molecule to emit photons of light.

Fluorogens may also be used to label proteins. Examples of fluorogensinclude fluorescein and derivatives, phycoerythrin, allo-phycocyanin,phycocyanin, rhodamine, and Texas Red. The fluorogens are generallydetected by a fluorescence detector.

The protein can alternatively be labeled with a chromogen to provide anenzyme or affinity label. For example, the protein can be biotinylatedso that it can be utilized in a biotin-avidin reaction, which may alsobe coupled to a label such as an enzyme or fluorogen. For example, theprotein can be labeled with peroxidase, alkaline phosphatase or otherenzymes giving a chromogenic or fluorogenic reaction upon addition ofsubstrate. Additives such as 5-amino-2,3-dihydro-1,4-phthalaz-inedione(also known as Luminol™) (Sigma Chemical Company, St. Louis, Mo.) andrate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol)(Sigma Chemical Company, St. Louis, Mo.) can be used to amplify enzymessuch as horseradish peroxidase through a luminescent reaction; andluminogeneic or fluorogenic dioxetane derivatives of enzyme substratescan also be used. Such labels can be detected using enzyme-linkedimmunoassays (ELISA) or by detecting a color change with the aid of aspectrophotometer. In addition, proteins may be labeled with colloidalgold for use in immunoelectron microscopy in accordance with methodswell known to those skilled in the art.

The location of a ligand in cells can be determined by labeling anantibody as described above and detecting the label in accordance withmethods well known to those skilled in the art, such asimmunofluorescence microscopy using procedures such as those describedby Warren and Nelson, Mol. Cell. Biol. 7: 1326-1337 (1987).

Screening Methods

The ClfB, SdrC, SdrD and SdrE proteins, or fragments thereof, such asconsensus or variable amino acid motifs, are useful in a method forscreening materials to identify substances that inhibit S. aureus hostcell binding interactions with the extracellular matrix. In accordancewith the method for screening, the substance of interest is combinedwith one or more of the ClfB, SdrC, SdrD, or SdrE proteins, or fragmentsthereof, such as consensus or variable sequence amino acid motifpeptides, and the degree of binding of the molecule to the extracellularmatrix is measured or observed. If the presence of the substance resultsin the inhibition of binding, then the substance may be useful forinhibiting S. aureus in vivo or in vitro. The method could similarly beused to identify substances that promote S. aureus interactions with theextracellular matrix.

The method is particularly useful for identifying substances havingbacteriostatic or bacteriocidal properties.

For example, to screen for S. aureus agonists or antagonists, asynthetic reaction mixture, a cellular compartment (such as a membrane,cell envelope or cell wall) containing one or more of the ClfB, SdrC,SdrD, SdrE proteins, or fragments thereof, such as consensus or variablesequence amino acid motifs, and a labeled substrate or ligand of theprotein is incubated in the presence of a substance under investigation.The ability of the substance to agonize or antagonize the protein isshown by a decrease in the binding of the labeled ligand or decreasedformation of substrate product. Substances that bind well and increasethe rate of product formation from substrate are agonists. Detection ofthe rate or level of formation of product from substrate may be enhancedby use of a reporter system, such as a calorimetric labeled substrateconverted to product, a reporter gene that is responsive to changes inClfB, SdrC, SdrD, SdrE or consensus or variable amino acid sequencemotifs' nucleic acid or protein activity, and binding assays known tothose skilled in the art. Competitive inhibition assays can also beused.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to ClfB, SdrC, SdrD, SdrE orconsensus or variable sequence amino acid motifs' nucleic acid moleculesor proteins and thereby inhibit their activity or bind to a bindingmolecule (such as fibrinogen) to prevent the binding of the ClfB, SdrC,SdrD, SdrE or consensus or variable sequence amino acid motifs' nucleicacid molecules or proteins to the binding molecule. For example, acompound that inhibits ClfB, SdrC, SdrD, SdrE or consensus or variablesequence amino acid motifs' activity may be a small molecule that bindsto and occupies the binding site of the ClfB, SdrC, SdrD, SdrE orconsensus or variable sequence amino acid motif peptide or protein,thereby preventing binding to cellular binding molecules. Examples ofsmall molecules include, but are not limited to, small organicmolecules, peptides or peptide-like molecules. Other potentialantagonists include antisense molecules. Preferred antagonists includecompounds related to and variants or derivatives of ClfB, SdrC, SdrD,SdrE or consensus or variable sequence amino acid motif peptides orproteins.

The nucleic acid molecules described herein may also be used to screencompounds for antibacterial activity.

Therapeutic Applications

In addition to the therapeutic compositions and methods described above,the ClfB, SdrC, SdrD, SdrE or consensus or variable amino acid motifs,peptides or proteins, nucleic acid molecules or antibodies are usefulfor interfering with the initial physical interaction between a pathogenand mammalian host responsible for infection, to mammalian extracellularmatrix proteins on indwelling devices or to extracellular matrixproteins in wounds they are further useful to block ClfB, SdrC, SdrD,SdrE, or active fragments thereof, including consensus or variable aminoacid motifs, peptide or protein-mediated mammalian cell invasion. Inaddition, these molecules are useful to mediate tissue damage and toblock the normal progression of pathogenesis in infections.

S. aureus Detection Kit

The invention further contemplates a kit containing one or more ClfB,SdrC, SdrD, SdrE proteins, peptides, or active fragments thereof,including consensus or variable amino acid motif-encoding nucleic acidprobes. These probes can be used for the detection of S. aureus or S.aureus extracellular matrix-binding proteins in a sample. Such a kit canalso contain the appropriate reagents for hybridizing the probe to thesample and detecting bound probe.

In an alternative embodiment, the kit contains one or more ClfB, SdrC,SdrD, or SdrE proteins, peptides or consensus or variable amino acidmotif-specific antibodies, which can be used for the detection of S.aureus organisms or S. aureus extracellular matrix-binding proteins in asample.

In yet another embodiment, the kit contains one or more ClfB, SdrC, SdrDor SdrE-proteins, or active fragments thereof, such as the consensus orvariable sequence amino acid motifs, which can be used for the detectionof S. aureus organisms or S. aureus extracellular matrix-bindingantibodies in a sample.

The kits described herein may additionally contain equipment for safelyobtaining the sample, a vessel for containing the reagents, a timingmeans, a buffer for diluting the sample, and a calorimeter,reflectometer, or standard against which a color change may be measured.

In a preferred embodiment, the reagents, including the protein orantibody, are lyophilized, most preferably in a single vessel. Additionof aqueous sample to the vessel results in solubilization of thelyophilized reagents, causing them to react. Most preferably, thereagents are sequentially lyophilized in a single container, inaccordance with methods well known to those skilled in the art thatminimize reaction by the reagents prior to addition of the sample.

EXAMPLES

The present invention is further illustrated by the followingnon-limiting examples, which are not to be construed in any way asimposing limitations upon the scope thereof. On the contrary, it is tobe clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the spirit of the present invention.

Example 1 Gene Cloning, Sequencing and Expression

A fibrinogen-binding protein gene, designated clfB, was isolated, clonedand sequenced as follows:

Bacterial Strains and Growth Conditions

The E. coli and S. aureus strains used for the cloning and sequencing ofclfB are listed in Table 1, below. Escherichia coli was routinely grownon L-broth or agar. S. aureus was routinely grown on trypticase soybroth (Oxoid) or agar. The following antibiotics were incorporated intomedia where appropriate: ampicillin (Ap), 100 μg/ml; tetracycline (Tc),2 μg/ml; chloramphenicol (Cm), 5 μg/ml; erythromycin (Em) 10 μg/ml.

Bacterial Relevant properties/ Strain Genotype Use in present studySource/reference E. Coli C600 F⁻, lacY1, leuB6, supE44, Propagation oflambda Appleyard, Genetics thi-1, thr-1, tonA21 recombinants 39: 440-452(1954) DH5α F⁻, ø80dlacZM15, deoR, Recombination deficient, host Hanahanet al, J. Mol. endA1, gyrA96, hsdR17, strain for plasmids and for DNABiol. 166: 557-580 (r_(k−), m_(k+)), (lacZYA-argF) sequencing (1983)U169, recA1, relA1, supE44, thi-1 JM101 supE, thi-1, (lac-proAB), Hoststrain for plasmid bank and Stratagene [F′ traD36, proAB, for sequencing(La Jolla, CA) lac1^(q)ZM15] LE392 F⁻, (r_(k−), m_(k+)), galK3,Propagation of lambda Promega Corp. galT22, hsdR574, lacY1 recombinants(Madison, WI) or (lacIZY)6, metB1, supE44, supF58, trpR55 XL-1 Blue [F′proAB, lacI^(q)ZM15, Propagation of Tn10(tc^(r))], endA1, gyrA96,plasmids Stratagene hsdR17, lac, recA1, relA1, supE44, thi-1 S. aureusNewman Strong adherence to fibrinogen NCTC 8178; Duthie and Lorenz, J.Gen. Microbiol. 6: 95-107 (1952) DU5876 clfA2::Tn917, Em_(r) McDevit etal., Mol. Microbiol. 11: 237-248 (1994) DU5943 clfB::Tc_(r), Tc_(r)described herein DU5944 clfAclfB, Em_(r), Tc_(r) described herein DU5874spa::Tc_(r) Protein A-defective mutant of NewmanMcDevitt et al., Mol.Microbiol. 16: 895-907 (1995) Δ map McDevitt, unpublished 8325-4 NCTC8325 cured of prophages Novick, Virology 33: 155-166 (1967) ISP546agr::Tn551 8325-4 agrBrown and Pattee, Infect. Immun. 30: 36-42 (1980)RN4220 Restriction deficient derivative Kreiswirth et al., of 8325-4Nature 305: 709-712 (1983) V8 Classic V8 protease producer, ATCC 27733produces PV leukocidin Cowan 1 Classic protein A producer, ATCC 12598adheres well to fibrinogen and fibronectin RN4282 TSST-1 producerKreiswirth et al., 1983 (as 3-14) Phillips Collagen binding strain Pattiet al., Infect. Immun. 62: 152-161 (1994) V13 Septicaemia isolateO′Reilly et al., Mol. Microbiol. 4: 1947-1955 (1990) GH13 Methicillinresistant Poston and Li Saw Hee, J. Med. Microbiol. 34: 193-201 (1991)P1 Rabbit virulent strain Sherertz et al., J. Infect. Dis. 167: 98-106(1993) M60 Bovine mastitis isolate Anderson, Zentralbl BakteriolParasitenkd Infektionskr Hyg Abt. 1 Orig Reihe A 5(Suppl.): 783-790(1976)

DNA Manipulation

Unless otherwise specified, DNA manipulations were done according tostandard methods as described by Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY. New York, John Wiley and Sons (1987) and Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. Cold SpringHarbour, N.Y., Cold Spring Harbour Laboratory Press (1989). Enzymes forDNA manipulation were obtained from New England Biolabs (Beverly, Mass.)and Promega (Madison, Wis.), and used as directed by the manufacturer.Genomic DNA from S. aureus Newman was prepared according to methods ofMuller et al., Infect. Immun. 61:551-558 (1993). Smaller scalepreparations were made by lysing cells in phosphate buffered saline(PBS) containing 12 μg/ml lysostaphin and 20 mM EDTA(ethylenediaminetetraacetic acid), followed by protease K treatment (500μg/ml in 1% SDS) for 1 hour at 60° C., extraction with phenol andchloroform, and dialysis against 10 mM Tris HCl, pH 8.0, 1 mM EDTA.Plasmid DNA was prepared from S. aureus according to the method ofVriesema et al., Appl. Environ. Microbiol. 62:3527-3529 (1996). E. coliplasmid DNA for use in polymerase chain reaction (PCR) and sequencingwas routinely made by the modified alkaline lysis method of Felicielloand Chinali, Anal. Biochem. 212:394-401 (1993), and occasionally bylarge scale isolation and dye-buoyant density centrifugation. Screeningof E. coli transformants for chimeric plasmids was routinely done by therapid colony lysis procedure of Le Gouill and Dery, Nucl. Acids Res.19:6655 (1994).

Cloning of Repeat-Containing Loci

A genomic library of S. aureus Newman was constructed in theLambdaGEM-12 replacement vector (obtained as prepared XhoI half-sitearms from Promega Madison, Wis.)) according to the manufacturer'sinstructions. Oligonucleotide probes specific for regions A and R of S.aureus Newman were made by polymerase chain amplification of theseregions from the cloned gene on pCF14, as described by McDevitt andFoster, Microbiology 141:937-943 (1995), and random-primer labeled with[alpha-³²P]dATP using the Promega Prime-a-Gene™ kit (Promega). The bankwas screened by Southern blotting, using an overnight hybridizationtemperature of 65° C. Selected clones were single plaque purified twice,and plate-lysate stocks made for storage and for inoculation of liquidcultures for the large-scale preparation of phage for DNA isolation.

A 3.87-kb HindIII fragment containing homology to region R DNA wascloned from the genome of S. aureus. HindIII-cleaved genomic DNA in therange of 3-4 kb was excised from an agarose gel, purified, and ligatedto the pBluescript cloning vector. Plasmids were transformed into E.coli JM101 and identification of a recombinant E. coli containing aregion R DNA insert was identified by PCR screening. PCR products weregenerated using primers specific for region R DNA. Individual colonieswithin a pool producing a positive PCR reaction were then analyzed fortheir potential to generate a PCR product. One transformant, pC1, wasidentified and found to contain the 3.87-kb fragment with homology toregion R.

DNA Sequencing

The DNA sequence of clfB was obtained from pA1-1EX, a plasmid containinga fragment subcloned from recombinant phage A1-1 into pGEM 7Z (f)+.Nested deletions were made using the Erase-a-Base™ Kit (Promega). TheFlash Dye Primer Sequencing Kit (Genpak) was used for sequencing in aModel 373A sequencing system (Applied Biosystems, Foster City, Calif.).Confirmatory sequencing in the forward direction was carried out. Doublestranded sequencing of sdrD and sdrE was done on the subclones pEJ1,pEJ2 and pEJ3, containing fragments subcloned from recombinant phageA6-2 in pGEM 7Z (f)+, by nested deletions and primer walking. Automatedsequence analysis of sdrC and the 5′ end of sdrD on plasmid clone pC1was performed. Sequence analysis was performed on both strands by primerextension to known sequences.

Screening of S. aureus Strains for clfB Homologues

A probe specific for the region A-encoding portion of clfB was made byexcising a 614 by internal AccI fragment from pA1-1EX, purifying from anagarose gel using the GENECLEAN II™ kit (BIO 101 Inc., La Jolla,Calif.), and labeling with [alpha-³²P]dATP as described in FIG. 2. Aprobe was similarly made to distal regions of the gene (encoding regionR, the wall and membrane-spanning regions, and about 100 by ofdownstream DNA), using a 1.2 kb BamHI fragment from pA1-1EX. HindIIIdigests of genomic DNA from a panel of strains were Southern blotted andscreened using these probes.

Expression of clfB Region A

Region A (encoding residues S45 to N542) of clfB was amplified frompA1-1EX by PCR using the following primers:

(SEQ ID NO: 15) Forward: 5′ CGAGGATCCTCAGGACAATCGAACGATACAACG 3′ (SEQ IDNO: 16) Reverse: CGAGGTACCATTTACTGCTGAATCACC 3′.

Cleavage sites for BamHI and KpnI (underlined) were appended to the 5′ends of the respective primers to introduce these sites into the productand facilitate its cloning into expression vectors. The forward primerwas subsequently found to include a single base mismatch (G,underlined), changing an E codon to a G codon. Reaction mixtures (50 μl)contained 2 mM dNTPs, 1.5 mM MgCl.sub.2, 1 ng pA1-1EX, 50 nM primers and1.25 U Taq polymerase in standard Promega (Madison, Wis.) Taq reactionbuffer. Amplification proceeded in a Perkin Elmer Cetus (Foster City,Calif.) thermocycler with an initial denaturation at 94° C. for 4minutes, followed by 30 cycles with denaturation at 94.degree. C. for 1minute, annealing at 50° C., and extension at 72° C. for 1.5 minutes,with minimum heating and cooling between steps. The final extension wasfor 10 minutes. A single product was obtained, which was purified usingthe Wizard™ PCR purification kit (Promega). The product was initiallycloned into the His-tag expression vector pQE30. However, becausehigh-level expression was not obtained in this system, the product wasrecloned into an alternative vector, the GST fusion vector pGEX-KG,between the BamHI and HindIII sites. The recombinant protein wasrecovered from lysates by affinity chromatography onglutathione-sepharose (GST Gene Fusion System™ Pharmacia, Piscataway,N.J.) and from the glutathione-S-transferase fusion partner by thrombincleavage.

Cloning of Repeat Carrying Loci

A library of S. aureus Newman genomic DNA was made using the replacementlambda vector LambdaGEM™-12. About 10 000 plaques were screened usingthe region R-specific probe. Of the 60 positive plaques retained, 26were purified and counter-screened with a clfA region A-specific probe.One plaque hybridized with the latter, indicating that it contained theclfA gene; of the remaining, non-hybridizing plaques, three wereselected at random, and the DNA isolated. The DNA was cut with severalrestriction enzymes and analyzed by Southern blotting using the region Rprobe. Clones A1-1 and A2-3 appeared to contain overlapping sequences.Restriction mapping and Southern blotting indicated that these clonescontained a single region R homologue. Clone A6-2 was found to containthree region R homologues, since cleavage with EcoRV yielded threefragments hybridizing to the region R probe.

Clone A1-1 was chosen for more detailed study, as the hybridizingfragment was slightly longer than in clone A2-3. A 7.4 kb EcoRI fragmentcontaining the repeat region was subcloned from lambda clone A1-1 intoplasmid pGEM 7Z f(+) to generate plasmid pA1-1E. This insert was reducedto approximately 3 kb by excision of a 4.4 kb XbaI segment to formpA1-1EX as shown in FIGS. 2 and 3.

Clone A6-2 was restriction mapped and fragments subcloned into plasmidvectors for sequencing as shown in FIG. 4. Southern blotting with theregion R probe and preliminary sequencing suggested that there werethree tandemly arrayed genes carrying region R encoding sequences. OnA6-2 there were two complete ORFs, sdrD and sdrE, and one incompleteORF, sdrC.

The two complete ORFs were sequenced on fragments subcloned from lambdaA6-2 into plasmid vectors pGEM7Z f(+) (subclones pEJ1 and pEJ2) andpBluescript KS+(subclone pEJ3). sdrC was cloned separately from S.aureus genomic DNA. A 3.87-kb HindIII fragment of strain Newman wascloned directly into plasmid pBluescript KS+, generating clone pC1 (FIG.4). This clone, containing a region R DNA insert, was identified by PCRscreening. The sequence of sdrC and the 162 by at the 5′ end of sdrDwere determined from pC1.

Plasmid pA1-1EX, carrying the clfB gene, was deposited at the NationalCollections of Industrial and Marine Bacteria on Oct. 13, 1997 under theAccession No. 40903. Plasmid pC1, carrying the gene for sdrC, wasdeposited at the National Collections of Industrial and Marine Bacteriaunder the Accession No. NCIMB 40902 on Oct. 13, 1997 and a recombinantlambda phage A6-2, carrying the sdrD and sdrE genes, was deposited atthe NCIMB on Oct. 13, 1997 under the Accession No. NCIMB 40904. Alldeposits comply with the terms of the Budapest Treaty.

Features of ClfB

The translated open reading frame (ORF) contained within pA1-1EX isshown in FIG. 5. The ORF shows features reminiscent of secreted proteinsof Gram positive cocci. Although the entire ORF is shown in FIG. 5, thestart codon is unlikely to be the N codon. There is no ATG codon at the5′ end of the ORF. However, GTG and TTG are occasionally used astranslational start codons in S. aureus, although methionine is theactual amino acid residue inserted, e.g., the fibronectin bindingproteins (GTG), and protein A (TTG). The first TTG codon (L) may well bethe initiation codon, as a possible ribosome binding site, GGAG, issuitably located upstream, starting at position-12. The N-terminal 44amino acid residue region thus predicted has properties similar tosignal sequences of secreted proteins of Gram positive cocci, i.e., aninitial stretch of 19 mostly polar residues, with an overall positivecharge, followed by 18 neutral residues with a high content ofhydrophobic residues, and finally a short stretch of mainly polarresidues with a good consensus cleavage site, AQA-S.

If the above prediction of the signal sequence is correct, region A ofClfB is 498 residues long, and shows 26.3% residue identity with theequivalent region in ClfA, or 44.4% homology when conservativesubstitutions are included. The most marked stretch of amino acidsimilarity between ClfA and ClfB occurs between residues 314-329 (ClfA)and 304-319 (ClfB), with 7 identical and 5 conserved residues. In ClfA,the stretch overlaps the C-terminal half of a putative Ca²⁺ bindingloop, EF hand I, required for fibrinogen binding as shown in FIG. 6. Thesequence DYSNS (SEQ ID NO: 11), which obeys the consensus for theN-terminal moiety of a MIDAS motif, occurs a short distance upstream.Accordingly, the downstream sequence was inspected for D and T residuesto complete the motif. D and T occur frequently throughout the protein,and T 339 is suitably located, 63 residues downstream. However, theconsensus would require a D residue 14-23 residues downstream from theT, and in the present case, the nearest D residues are 9 or 28 residuesaway (D 348 and D 367).

At the C-terminal end of region A, a prominent proline-rich regionoccurs (21/42 residues are P; as shown in FIG. 5). There is a 14-residuerepeat within this sequence. The DNA encoding the P-rich repeats ishighly conserved. Of the three base substitutions, only one results inan amino acid replacement, a conservative substitution of S for T.

Region R is somewhat shorter in clfB than in clfA (272 residues insteadof 308). The region R encoding sequence comprises the 18-bp consensusrepeat observed in the equivalent part of clfA.

Following region R is a short stretch of predominantly hydrophilicresidues, containing the distinctive LPETG (SEQ ID NO: 22) motif nearits C-terminal end, presumably the cell sorting signal. The C-terminalregion of the predicted protein shows strong homology with thecorresponding region in ClfA, with an initial stretch of mostlyhydrophobic residues and a final stretch rich in positively chargedresidues, reminiscent of membrane spanning and anchoring domains,respectively. The general organization of ClfA and ClfB is compared inFIG. 1.

A putative transcription termination signal occurs 3′ to clfB. No openreading frames occur within 260 by 5′ or 200 by 3′, suggesting that thegene is not part of an operon.

Features of SdrC, SdrD and SdrE

The DNA sequences and the translated amino acid sequences of sdrC, sdrDand sdrE are shown in FIGS. 7, 8 and 9. Each predicted protein has aputative signal sequence, an approximately 500 residue “region A” withlimited homology to region A of ClfA (see FIG. 10), variable numbers ofB repeats, an SD repeat containing region R, an LPXTG (SEQ ID NO: 14)cell wall sorting motif, a hydrophilic membrane anchor, and positivelycharged residues at the extreme C terminus.

The organization of the five region R containing proteins is shown inFIG. 10. The A regions of SdrC, SdrD and SdrE have limited sequencesimilarity to each other and to those of ClfA and ClfB as shown in FIG.11. Alignments of those sequences more strongly conserved between allfive proteins are shown in FIG. 12. The consensus motif TYTFTDYVD (SEQID NO: 18) overlaps the EF hand 1 motif of ClfA (alignment 2, FIG. 12).This region of ClfA has been shown to be of crucial significance in itsligand (fibrinogen) binding activity as described by O′Connell et al.,J. Biol. Chem., 273:6821-6829 (1998), and may also be of importance inthe biological activity of the new proteins.

The three proteins SdrC, SdrD and SdrE form a separate subgroup ofregion R containing proteins: in addition to regions R and A theycontain variable numbers of B repeats, located between region A andregion R. The B repeats are 110-113 amino acids long and showconsiderable similarity (alignment 5, FIG. 12). The repeats SdrC B2,SdrD B5 and SdrE B3 adjacent to region R are 93-95% identical. There isa strongly conserved EF hand near the N-terminal end of each repeat.

clfB Homologues in other S. aureus Strains

Nine strains of S. aureus were screened for the clfB gene by Southernblotting. Genomic DNA was cut to completion with HindIII, and probedwith an internal 0.6 kb AccI fragment of the region A coding sequence ofclfB, shown in FIG. 2. The probe recognized a single HindIII fragmentvarying from 2 to 3 kb in length in all nine strains, indicating thateach possesses a single clfB allele. A probe made from the region R anddistal regions of clfB recognized an identical band in all strains,indicating that the clfB homologues in other strains also contain regionR.

Expression of clfB

The portion of clfB encoding region A was amplified by PCR using primersincorporating suitable 5′ restriction sites, and cloned into the E. coliexpression vector pGEX-KG. A protein of 94.3 kDa was detected in lysatesof induced bacteria. The GST-ClfB fusion protein was immobilized on aglutathione sepharose affinity column, cleaved with thrombin, andexamined by SDS-PAGE. The predominant band was 42 kDa, whereas thecalculated molecular weight of region A is 54 kDa. This protein was usedto raise antibody in rabbits, to probe Western blots of cell lysatesmade from strain Newman grown under a variety of conditions, asdescribed below. The antibody failed to detect any antigens in lysatesmade from plate cultures, statically grown broth cultures, orshake-flask cultures grown to stationary phase. A single 124-kDa bandwas detected in lysates made from exponential phase shake-flask culturesof strain Newman and derivatives. If it is assumed that processingremoves the signal sequence and the C-terminal portion of the proteinfrom the last G of the LPETG (SEQ ID NO: 22), the predicted molecularweight of ClfB is 88.3 kDa. In a time-course of ClfB production by ashake-flask culture of strain Newman, the ClfB protein was most abundantin the early exponential phase and showed a sharp decline toward the endof exponential phase, after which levels became undetectable. Theresults of the time-course study are shown in FIG. 13.

Example 2 Production of Anti-ClfB Serum

Antibodies to recombinant region A were raised in two young New Zealandwhite rabbits (2 kg) showing no prior reaction with E. coli or S. aureusantigens in Western blots. Injections, given subcutaneously, contained25 μg of the antigen, diluted to 500 ml in phosphate buffered saline(PBS) emulsified with an equal volume of adjuvant. The initial injectioncontained Freund's complete adjuvant; the two to three subsequentinjections, given at two-week intervals, contained Freund's incompleteadjuvant. When the response to the recombinant protein was judgedadequate, the rabbits were bled, serum recovered, and total IgG purifiedby affinity chromatography on protein A sepharose (Sigma Chemical Co.,St. Louis, Mo.).

SDS-PAGE and Western Blotting

Samples were analyzed by SDS-PAGE in 10 or 12% acrylamide gels. Isolatedproteins and E. coli cell lysates were prepared for electrophoresis byboiling for five minutes in denaturation buffer. For S. aureus, cellswere suspended to an OD₆₀₀ of 40 units in 100 mM PBS containing 10 mMEDTA. To each 500 μl sample, 40 μl protease inhibitors (Complete™cocktail, Boehringer Mannheim, Indianapolis, Ind.), 5 μl each of DNAseand RNAse (from 10 mg/ml stocks, Sigma Chemical Co.), and 60 μl of a 2mg/ml lysostaphin stock (Ambicin L™ recombinant lysostaphin, AppliedMicrobiology Inc., Tarrytown, N.Y.) were then added and the suspensionincubated in a 37° C. water bath until it cleared. The samples were thenprocessed as usual. Gels were stained with Coomassie blue or transferredto Nytran™ membrane by Western blotting in the Bio-Rad Semidry™ system(Bio-Rad Laboratories, Richmond, Calif.). For detection of native ClfBin S. aureus, blots were processed using the BM ChemiluminescenceDetection System™ (POD) of Boehringer Mannheim, according to themanufacturer's instructions. Primary anti-ClfB antibody was used at a1/1000 dilution, for a two hour incubation at room temperature. ProteinA conjugated with horse radish peroxidase (Sigma Chemical Co.) was usedto detect bound antibody, diluted 1/2000 for a one hour incubation atroom temperature. Blots requiring less sensitivity were treated in asimilar way, except that 5% skim milk was used as a blocking agent, andthe blots were developed using chloronaphthol and hydrogen peroxide.

To determine whether ClfB is cell wall-associated, whole cells from anexponential phase culture were treated with lysostaphin in buffersupplemented with 30% raffinose to stabilize the protoplasts. Theprotoplasts were harvested, and the protoplasts and supernatant analyzedseparately by Western blotting. ClfB protein was detected only in thesupernatant, indicating that all ClfB was cross-linked to thepeptidoglycan, and could be released by lysostaphin without disruptionof the protoplast.

ClfB expression was enhanced by growth in rich media, such as tryptonesoy broth or brain heart infusion.

Several S. aureus strains known to contain clfB alleles were screenedfor ClfB production by Western blotting. Cultures were harvested inearly exponential phase to maximize expression. Of the nine strainsexamined, 83254, RN4282, and V13 expressed immunoreactive antigens ofsimilar size and intensity to that of Newman, whereas strains GH13 andP1 had very weak bands of this size. Strains P1, Cowan and M60 expressedsmaller immunoreactive antigens which may be degradation products.Strains V8 and Phillips expressed no detectable ClfB protein. StrainRN4220, which was derived from 8325-4, expressed exceptionally highlevels of ClfB.

Example 3 Immunoassay for ClfB using Biotinylated Recombinant ClfBRegion A

The DNA encoding region A of clfB (encoding residues S45 to N542) wasamplified from genomic DNA of S. aureus Newman using the followingprimers:

(SEQ ID NO: 17) Forward: 5′ CGAAAGCTTGTCAGAACAATCGAACGATACAACG 3′ (SEQID NO: 16) Reverse: 5′ CGAGGATCCATTTACTGCTGAATCACC 3′

Cleavage sites for HindIII and BamHI (underlined) were appended to the5′ ends of the respective primers to facilitate cloning of the productinto the His-tag expression vector pV4. Cloning employed E. coli JM101as a host strain. The recombinant region A was purified by nickelaffinity chromatography.

Enzyme Linked Immunosorbent Assay (ELISA)

Immulon 1™ plates (Dynatech™, Dynal, Inc., Great Neck, N.Y.) were coatedovernight with 100 μl of 10 μg/ml human fibrinogen (Chromogenix). Theywere then blocked with 200 μl of 5 mg/ml bovine serum albumin (BSA) forone hour. The plates were then incubated for three hours with 100 μlbiotinylated ClfB (His-tag recombinant region A) diluted to 0.1-10μg/ml. They were then given three five-minute washes with PBS containing0.02% Tween 20 and 1 mg/ml BSA. The plates were then incubated for onehour with 100 μl of a 1/10 000 dilution of streptavidin conjugated withalkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.), andwashed as before. The plates were then developed for 30 minutes at 37°C. with 100 μl of 1 M diethanolamine, pH 9.8, containing 1 mg/mlp-nitrophenyl phosphate (Sigma Chemical Co.). Plates coated with BSAonly were used as negative controls. The absorbance was measured at 405nm.

Western Affinity Blotting

A 20. μg quantity of human fibrinogen (Chromogenix) was subjected toSDS-PAGE on a 15% acrylamide gel for two hours. Proteins weretransferred to nitrocellulose at 100 V for two hours. The membrane wasblocked overnight in PBS containing 10% nonfat dry milk. The blot wasthen incubated with 2.5 μg/ml biotinylated ClfB (His-tag recombinantregion A) for one hour with shaking, the biotinylation being performedwith EZ link-sulfo-NHS-LC-Biotin™ (Pierce, Rockford, Ill.). The blot wasthen given three five-minute washes in PBS containing 0.1% Tween 20. Theblot was then incubated for one hour with avidin conjugated withhorseradish peroxidase (Boehringer Mannheim) at a 1/200,000 dilution.The blot was then washed as before, and developed using the enhancedchemiluminescence system of Amersham (Little Chalfont, Bucks, UK). Theband profile was compared with that obtained by subjecting fibrinogen toSDS-PAGE and Coomassie Blue staining.

In a Western affinity blot, in which biotinylated purified ClfB region Awas used to probe blotted fibrinogen, a comparison with a lane ofstained fibrinogen indicated that ClfB bound the alpha and beta-chainsof fibrinogen. No bands were seen when ClfB was omitted. This experimentshows an important difference with ClfA, which is known to bind to thegamma-chain of fibrinogen.

Example 4 Mutagenesis of clfB

An insertion mutation in clfB was created by introducing a fragmentcontaining a Tc resistance marker into the middle of the gene on pA1-1EXas shown in FIG. 3. The 2.35-kb HindIII fragment from pT181 was filledin with Klenow enzyme, and blunt-end ligated into the HpaI site ofpA1-1EX. Plasmid pTS2, with temperature sensitive replication and aCm.sup.r marker, was cloned into this construct at the SmaI site bycleaving with AvaI. This cloning step was carried out in E. coli, andtransformants were selected on Ap and incubated at 30° C. to avoidselection of revertants to temperature independence. The plasmid wasthen purified and transformed into S. aureus RN4220 by electroporationand Tc^(r) transformants selected at 30° C. Five independent brothcultures grown at 30° C. were diluted 1/100 in fresh medium withoutantibiotics, and grown at 42° C. for six hours or 18 hours. The cultureswere then diluted 1/100 and incubated at 42° C. for another time period.Six such dilutions and incubations were made, by which time Tcresistance had declined to approximately 1/1000 colony forming units(CFU). The cultures were then diluted to give approximately 100 CFU perplate on medium containing Tc, and incubated overnight at 37° C.Colonies which were Tc^(r) but Cm^(s) were presumed to have undergone adouble crossover event between the plasmid and host genome, leading toreplacement of the wild-type gene with the mutated one, with subsequentloss of the plasmid. Five hundred colonies were screened per culture.Eleven presumptive mutants were isolated from four of the five cultures.Four representative mutants were selected and genomic DNA isolated.Mutant DU5944, deficient in both clfA and clfB, was constructed bytransducing clfA2::Tn917 from strain DU5876 into clfB mutant DU5943,selecting for Em^(r).

To determine whether mutations known to affect exoprotein expressioninfluenced clfB, strain 8325-4 and the agr mutant ISP546 were compared.No significant differences in the level or dynamics of ClfB expressionwere noted.

To determine the role of ClfB in bacteria-fibrinogen interactions, aclfB mutant of strain Newman was constructed by allele replacement asshown in FIG. 2. Genomic DNA of the mutant was digested with BamHI andsubjected to Southern blotting with a labeled 1.3 kb HpaI fragment fromplasmid pA1-1E containing the 5′ half of clfB and about 150 by ofupstream sequence. A single band hybridized in each case, but asexpected, the band was 2.35 kb longer in the mutant than in thewild-type. The mutation was initially isolated in RN4220 and thentransduced into strain Newman, forming strain DU5943.

Overexpression of ClfB and Complementation of clfB Mutation

Overproduction of ClfB was enabled by subcloning a SmaI fragmentcontaining the clfB gene and 500 by of upstream DNA from pA1-1E into thehigh copy number shuttle plasmid pCU1. The construct was thentransformed into strain RN4220 and transduced into strain NewmanTransductants were selected on Cm. Southern and Western blottingconfirmed that the high copy number was maintained in strain Newman, andthat ClfB was produced at higher than wild-type levels, indicating thatthe upstream DNA contained the promoter necessary for expression of theclfB gene. Transduction of the construct into clfB mutants restored ClfBsynthesis to higher than wild-type levels. The construct was alsotransduced into clfAclfB double mutants for use in complementationstudies.

To create a clfAclfB double mutant, a clfA::Tn917 mutation wastransferred by transduction from strain DU5876 into the clfB::Tc^(r)mutant DU5943, forming DU5944. The wild-type clfB gene was cloned intoshuttle plasmid pCU1 to give plasmid pA1-1EA, which was introduced intothe clfAclfB mutant by transduction to test complementation. Westernblotting with anti-ClfB serum showed that the ClfB protein was missingin mutant DU5943. It was expressed at a higher level than the wild-typein mutants carrying the complementing plasmid PA1-1EA, indicatingoverexpression of the protein due to gene dosage effect.

Example 5 ClfB Binding Assays

Clumping Assays

The role of ClfB in binding of S. aureus cells to soluble fibrinogen wasinvestigated in clumping assays. Clumping assays were carried out inSarstedt™ flat-bottomed multiwell test plates, using 50-μl volumes ofhuman fibrinogen (Calbiochem Corp. (San Diego, Calif.) plasminogenfree, >95% pure), diluted serially two-fold in PBS from a startingconcentration of 1 mg/ml. S. aureus cultures were washed once in PBS,resuspended to a final OD₆₀₀ of 6, and 20 μl added to each well. Controlwells contained PBS 30 only. The plates were agitated briskly for fiveminutes and visually examined for clumping. The clumping titer was thelowest concentration of fibrinogen at which clumping occurred. Theresults are set forth in Table 2, below. Results are the mean ofconcurrent duplicate assays.

TABLE 2 Clumping titres of S. aureus Newman and mutants from differentculture phases Clumping titer, μg/ml Stationary Strain fibrinogen phaseExponential phase Wild-type 0.98 0.98 clfA 3.91 >1000.00 clfB 1.95 0.98clfA clfB >1000.00 >1000.00 clfA clfB (pA1- 2.93 250.00 1EA; clfB⁺)

The clumping titers of clfA and clfB single mutants were very similar towild-type when exponential phase cultures were used. However, the doubleclfAclfB mutant failed to form clumps, even at the highest fibrinogenconcentration. In contrast, the double mutant carrying the wild-typeclfB gene on pA1-1EA formed clumps with almost the same avidity as thewild-type. These data show unambiguously that ClfB is a clumping factor.

The difference in clumping titer between the single mutants was muchgreater when stationary phase cultures were used, where only ClfA ispresent on cells. The wild-type strain and single clfB mutant hadidentical titers. The single clfA mutant failed to clump, and was thusindistinguishable from the double mutant. Interestingly, there was aslight restoration of clumping when the double mutant was complementedwith the overexpressed clfB gene. This probably reflects over expressionof the protein.

Plate Adherence Assays

To determine whether ClfB can promote bacterial attachment toimmobilized fibrinogen, strains were tested for fibrinogen binding in amicrotiter plate adherence assay. Binding of cells to fibrinogenimmobilized on plates was measured by the assay of Wolz et al., Infect.Immun. 64:3142-3147 (1996). Fibrinogen was diluted in carbonate buffer(15 mM Na₂CO₃, 35 mM NaHCO₃, 3.2 μM NaN₃, pH 9.6) and 100 μl used tocoat 96-well flat-bottomed ELISA plates (Immulon 4™ Dynatech) overnightat 4° C. Control wells contained carbonate buffer only. After washing in150 mM NaCl, 0.05% Tween 20™ surfactant, the plates were blocked for onehour at 37.degree. C. in 1% BSA, 0.05% Tween in PBS. After washing asbefore, 100 μl of a cell suspension (OD₆₀₀ of 0.4 in PBS) was added, andthe plates incubated for two hours at 37° C. After gentle washing byhand, adherent cells were fixed by adding 100 μl of 25% aqueousformaldehyde, and incubating at room temperature for at least 30minutes. The plates were then washed gently once more, stained withcrystal violet, washed again, and the plates read by ELISA reader at 570nm. To avoid inter-assay variation, experiments were designed so that asingle plate provided a complete set of results.

The pattern of adherence strongly reflected that obtained in clumpingassays (FIG. 15). Assays in which the concentration of cells was variedindicated that binding was approximately half the maximum value at acell density of 0.4 OD (except for the double mutant), and this celldensity was subsequently used routinely. Wild-type, clfA, clfB mutantsand clfAclfB (pA1-1EA) showed a fibrinogen concentration-dependentincrease in binding (FIG. 16). This increase was less marked for theclfB mutant (expressing ClfA) than for the clfA mutant (expressingClfB), suggesting that ClfB may be a less avid and/or abundant receptor.With stationary phase cells, the clfB mutant continued to behave likethe wild-type strain, whereas the clfA mutant bound much less avidly. Aswith clumping, adherence was slightly higher with the complementeddouble mutant, presumably due to a gene dosage effect.

The clumping and adherence assays show that ClfB mediates binding bothto soluble and immobilized fibrinogen, closely resembling the activityof ClfA.

The binding of increasing concentrations of biotinylated purified regionA from ClfA and ClfB to solid phase fibrinogen was compared in a directELISA. The results are shown in FIG. 14. The adherence profiles of thetwo proteins were very similar, especially at the lower concentrations.At the highest concentration, binding of ClfA was approximately 50%greater than that of ClfB. Neither protein bound to BSA.

Effect of Anti-ClfB Antibody on Bacterial Adherence to ImmobilizedFibrinogen

To study inhibition of fibrinogen binding by IgG, the cells used for theassay were preincubated with serial two-fold dilutions of purified IgGin PBS, starting with a concentration of 500 μg/ml. Preincubation wasfor two hours at 37° C. in Sarstedt™ multiwell test plates, and thecells were then transferred to ELISA plates coated with fibrinogen (2.5μg/ml) and blocked as before. The rest of the assay was as before.

Cells from exponential phase cultures of wild-type and mutant Newmanstrains were preincubated with increasing concentrations of purifiedanti-ClfB IgG, and adherence to plastic surfaces coated with 2.5 μg/mlfibrinogen examined. The results are shown in FIG. 17. Binding of theclfB mutant was not inhibited, and binding of wild-type cells was almostunaffected, even at the highest antibody concentration. However, bindingof the clfA mutant showed an IgG concentration-dependent decrease, withan IC₅₀ of 16 μg/ml. The double mutant carrying clfB on a complementingplasmid was also inhibited by the antibody, although the IC₅₀ was higher(50 μg/ml), presumably because more ClfB was being expressed on the cellsurface.

Effect of Divalent Cations on Bacterial Adherence to ImmobilizedFibrinogen

The effect of metal ions on fibrinogen binding was studied in a similarmanner, preincubating the cells with serial two-fold dilutions of MgCl₂,MnCl₂ or MgCl₂ in TBS (50 mM Tris HCl, pH 7.5, 150 mM NaCl), startingwith a concentration of 50 mM. TBS was used instead of PBS, which causesprecipitation of both calcium and manganese. Since the cells bound lesswell under these conditions, the starting cell concentration wasdoubled.

It is known that the interaction of ClfA and fibrinogen is inhibited byCa²⁺ and Mn²⁺, but not Mg²⁺ ions. The effect of divalent cations onClfB-promoted adherence to fibrinogen was thus tested. Preincubation ofexponential phase cells of the wild-type strain and the clf mutants withCaCl₂ inhibited binding to fibrinogen. Those strains expressing ClfBalone showed greater sensitivity than the mutant expressing ClfA alone(clfB). The IC₅₀ for the wild-type strain and the clfB mutant were 17and 14 mM, respectively, whereas for the clfA mutant and the clfB⁺complemented double mutant the IC₅₀ was 1.05 and 0.60 mM, respectively.MnCl₂ also inhibited attachment of the wild-type strain and mutants,with a stronger effect on strains expressing only clfB. The IC₅₀ for thewild-type and the clfB mutant was 3.3 and 6.4 mM, respectively, whereasfor the clfA mutant and the double mutant carrying clfB+ on acomplementing plasmid the IC₅₀ was 0.35 and 1.26 mM respectively. MgCl₂had no effect on binding below 12.5 mM.

Thus, clfB promoted adherence of bacteria to immobilized fibrinogen isinhibited by Ca²⁺ and Mn²⁺ at similar concentrations to ClfA-promotedadherence. However, the mechanisms are likely to be different since ClfBdoes not contain a homologue of EF hand I implicated in Ca²⁺ promotedmodulation of ClfA-fibrinogen interactions.

Platelet-Fibrin Clot Adherence Assay

Adherence to platelet-fibrin clots was measured using a modification ofan assay employed by Moreillon et al., Infect. Immun. 63: 4738-4743(1995). Fresh canine blood was collected on 10% sodium citrate buffer(Sigma Chemical Co.), and centrifuged at 3000×.g for 10 minutes at roomtemperature. The plasma fraction was removed and placed in a clean tube.Platelet-fibrin clots were made by mixing 0.5 ml volumes of plasma with0.1 ml volumes of 0.2 mM CaCl₂ in 35 mm petri dishes. Thrombin (0.1 mlof 500 U/ml Sigma bovine thrombin) was then added, mixed in quickly, andthe clots allowed to form. To measure bacterial adherence, 2 ml of PBScontaining 5×10³ cfu/ml of bacteria (from a BHI-grown exponential phaseculture) was added to each dish, and the dishes shaken for three minuteson an orbital shaker. The inoculum was drained off and the clots washedtwice for five minutes each with 2 ml of PBS. The clots were thenoverlaid with 3 ml of molten TSA, incubated for 15 hours at 37° C., andthe colonies counted. The bacterial suspension used as an inoculum wasspread on TSA plates to obtain a total viable count, and the percentageof bound inoculum calculated. Results represent means of 6-10 plates perstrain, and were analyzed statistically using the student's T test.

The clfB mutation reduced adherence when compared to the wild-typestrain Newman, as did the clfA mutation which was previously shown byMoreillon et al. to have significantly reduced adherence in this model.

Assay for Adherence to Haemodialysis Tubing

In order to demonstrate that ClfB could serve as an adhesin for S.aureus in biomaterial-related infections, explanted human haemodialysistubing was tested for promotion of bacterial adherence in vitro. Thetubing was coated with a complex mixture of host plasma proteinsincluding fibrinogen and fibronectin.

These experiments employed sections of haemodialysis tubing removed frompatients 3 to 3.5 hours after implantation. Cultures were grown for twohours with shaking. Results, showing means with SEM of threeexperiments, are shown in FIG. 19.

Assay for Adherence to Fibrinogen-Coated PMMA Coverslips

Adherence of S. aureus Newman and mutants to fibrinogen-coatedpolymethylmethacrylate (PMMA) coverslips was measured as described byGreene et al., Mol. Microbiol. 17:1143-1152 (1995), except that thecoverslips were coated with pure fibrinogen (1 μg/ml). Cultures for theassay were grown for two hours with shaking. Results, showing the meansand SEM of triplicate experiments, are shown in FIG. 18.

The pattern of adherence to the tubing segments resembled the pattern ofbinding seen for immobilized fibrinogen in a parallel assay foradherence to fibrinogen immobilized on PMMA coverslips. The single clfAmutants had slightly lower levels of adherence compared to the wild-typewhereas the double clfAclfB mutant was reduced to approximately 30% ofwild-type level. Complementation of the single clfB mutant with the clfBgene on pA1-1EA restored binding to greater than wild-type levels,whereas complementation of the double mutant with the same plasmidrestored binding only to the same level as the single clfA mutant.

Example 6 ClfB as a Virulence Factor in Experimental Endocarditis

Clumping factor A was shown to be a virulence factor promoting adherenceto damaged heart valves in the rat model of experimental endocarditis ofMoreillon et al., Infect. Immun. 63:4738-4743 (1995). Therefore, therole of ClfB in this infection was tested by comparing the infectionrate of a clfB mutant and the mutant carrying the complementing clfBplasmid. Rats were infected intravenously at an ID₆₀ with 5×10³ cfu. 61%of the wild-type control animals' valves were infected (n=13), whereasonly 30% of the clfB mutant infected animals were colonized (n=20). Incontrast 77% (n=9) of the complemented mutant became infected. Thisclearly shows that ClfB is an adhesin and potential virulence factor inthe endocarditis model.

Example 7 Generation of TYTFTDYVD (SEQ ID NO:18) Peptide Antibodies

The nanopeptide, TYTFTDYVD (SEQ ID NO:18), was synthesized in multipleantigen peptide format (MAP; Research Genetics, Inc., Huntsville, Ala.).The peptide was conjugated to KLH according to manufacturers' directions(Pierce). Two female New Zealand White rabbits were immunizedsubcutaneously with the KLH-TYTFTDYVD (SEQ ID NO:18) conjugateemulsified with Freund's Complete Adjuvant. The rabbits were boosted 3weeks later by subcutaneous injection of KLH-TYTFTDYVD (SEQ ID NO:18)adjuvanted with Freunds Incomplete. A third boost was administeredsubcutaneously with KLH-TYTFTDYVD (SEQ ID NO:18) in PBS. The animalswere analyzed for TYTFTDYVD (SEQ ID NO:18) specific antibodies 21 daysafter the final boost. For purification of antibodies, antisera wasdiluted 1:1 with Tris-HCl pH 8.0 and passed over a Protein A-Sepharose®column After sequential washes with Tris-HCl pH 8.0, 0.5 M sodiumchloride, the bound antibodies were eluted in 3.5 M MgCl₂, and dialyzedinto PBS.

Immulon-2 microtiter plates (Dynex Technologies, Chantilly, Va.) werecoated for 2 hr at room temperature with 1 μg ClfA, ClfB, or BSA. Theprotein coated plates were washed three times with PBS, 0.05% Tween 20and then blocked with PBS, 1% BSA. The blocked plates were washed threetimes with PBS, 0.05% Tween 20. Fifty μl of the purified rabbitKLH-TYTFTDYVD (SEQ ID NO:18) antibodies were serially diluted in PBS andadded to the microtiter plate and incubated at 25° C. on a rockerplatform. The wells were washed three times with PBS, 0.05% Tween 20 andthe secondary antibody was added to the wells and incubated for 1 hr atroom temperature. The secondary antibody was alkalinephosphatase-conjugated goat anti-rabbit IgG (Bio-Rad), diluted 3000-foldin PBS. ELISA plates were developed for 1 hr at 37° C. with 1 mg/mlp-nitrophenyl phosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgCl₂, pH9.8, and quantified at 405 nm on a Perkin Elmer HTS 7000 Bio-Assayreader.

The data is shown in FIG. 21. These data indicate that theanti-consensus sequence TYTFTDYVD (SEQ ID NO:18) antibodiessignificantly bind to ClfA and ClfB proteins, but not to the controlprotein, BSA.

Example 8 Passive Immunization with Rabbit CUB IgG

The DNA encoding region A of clfB (encoding residues S45 to N542) wasamplified from genomic DNA of S. aureus Newman using the followingprimers:

(SEQ ID NO: 17) Forward: 5′ CGAAAGCTTGTCAGAACAATCGAACGATACAACG 3′ (SEQID NO: 16) Reverse: 5′ CGAGGATCCATTTACTGCTGAATCACC 3′

Cleavage sites for HindIII and BamHI (underlined) were appended to the5′ ends of the respective primers to facilitate cloning of the productinto the His-tag expression vector pV4. Cloning employed E. coli JM101as a host strain. The recombinant region A was purified by nickelaffinity chromatography. Antibodies were raised in rabbits with thepurified recombinant A region according to standard procedures.Anti-ClfB A region IgG was purified by affinity chromatography on aProtein A sepharose column.

Twenty Swiss Webster mice (23-28 g) were used to determine if passiveimmunization with purified rabbit anti-ClfB A region IgG could preventinfection mediated by a methicillin resistant S. aureus.

Methicillin resistant S. aureus strain 601 was cultured on blood agarplates. A single colony was then inoculated into 10 mls of BHI broth andincubated at 37° C. overnight. The culture was diluted to a 1:100dilution, placed into 10 ml of fresh BHI and grown to an optical density(O.D.) of 1.5-2.0. The culture was then centrifuged and washed in1.times.PBS. The culture was re-suspended in 1×PBS containing 5% BSA and10% dimethyl sulfoxide (DMSO) and kept frozen at −20° C. The bacterialsolution was thawed, washed, diluted in PBS, and adjusted to theappropriate concentrations before dosing the mice.

The mice were divided into four treatment groups (5 mice per treatmentgroup). Mice were assigned to treatment groups as follows:

Antibody/Bacteria Dose CFU/mouse No. of Mice 1 Normal rabbit IgG/S.aureus 3.81 × 10⁷ 5 2 Normal rabbit IgG/S. aureus 7.62 × 10⁷ 5 3 Rabbitanti-ClfB IgG/S. aureus 3.81 × 10⁷ 5 4 Rabbit anti-ClfB IgG/S. aureus7.62 × 10⁷ 5

On day −1, ten mice were administered 10 mg rabbit anti-ClfB region AIgG and 10 mice were given 10 mg normal rabbit IgG. Both antibodies weregiven via intraperitoneal (i.p.) injection. On day 0, all mice wereinfected intravenously (i.v.) with either 3.81×10⁷ CFU S. aureus or7.62×10⁷ CFU S. aureus.

Systemic infection was measured by evaluation of body weight loss. Bodyweight loss is one of the primary parameters that is evaluated whencases of illness and injury are being assessed in mice. The body weightof each animal was recorded on Day −1 and every other day thereafter,including terminal sacrifice. The animals were weighed to the nearest0.1 gram.

Mice injected with normal rabbit IgG displayed a significantly largerweight loss at the end of the experiment compared to mice passivelyimmunized with rabbit anti-ClfB region A IgG (see table below). Inaddition, pathological evaluation of the mice at necropsy revealed agreater number of lesions and foci of infection in the kidneys from themice receiving normal rabbit IgG compared to the kidneys from mice thatwere immunized with anti-ClfB region A IgG

% Change in body weight (mean) Normal IgG/ Anti-ClfB IgG/ Normal IgG/Anti-ClfB IgG/ Day of S. aureus S. aureus S. aureus S. aureus Study 3.81× 10⁷ 3.81 × 10⁷ 7.62 × 10⁷ 7.62 × 10⁷ −1 0 0 0 0 1 2.9 3.6 3.9 5.8 3 105.1 8.5 8.2 5 8.3 1.5 8.0 6.6

Example 9 ClfB Region A Binds .alpha. and .beta. Chains of HumanFibrinogen

Human fibrinogen (20 μg; Chromogenix) was separated by SDS-PAGE on a 15%acrylamide gel for 2 hours. Proteins were transferred to nitrocelluloseat 100 V for 2 h. The membranes were blocked overnight in PBS containing10% non-fat dry milk and then incubated with 2.5 μg/ml biotinylated ClfBor ClfA region A protein for 1 h with shaking. They were then given 3×5mm washes with PBS containing 0.1% Tween 20 and incubated for 1 hr withavidin conjugated horseradish peroxidase (Boehringer Mannheim: 1:100,000dilution). The filters were washed as before and developed usingenhanced chemilluminescence (Amersham). The Western Blot (FIG. 22)illustrates the binding of biotinylated ClfA to the γ chain offibrinogen and the binding of biotinylated ClfB to the α and β chains offibrinogen.

Example 10 ClfB Region A Binds 75 kD and 50 kD Proteins from HumanRhabdomyosarcoma Cell Line

Human Rhabdomyosarcoma Cells were lysed with the SDS-PAGE running bufferand varying amounts (2-10 μl) of the protein lysate were separated bySDS-PAGE on a 15% acrylamide gel for 2 h. Proteins were transferred tonitrocellulose at 100 V for 2 h. The membranes were blocked overnight inPBS containing 10% non-fat dry milk and then incubated with 2.5 μg/mlbiotinylated ClfB or ClfA region A protein for 1 hr with constantshaking. They were then given 3.times.5 min washes with PBS containing0.1% Tween 20 and incubated for 1 hr with avidin conjugated horseradishperoxidase (Boehringer Mannheim; 1:100,000 dilution). The filters werewashed as before and developed using enhanced chemilluminescence(Amersham). Two major bands were seen at 50 kD and 75 kD that reactedwith the biotinylated ClfB region A protein.

1. An isolated protein wherein the protein has an amino acid sequencecomprising the sequence of SEQ ID NO:
 5. 2. An isolated protein encodedby a nucleic acid sequence comprising the sequence of SEQ ID NO:
 6. 3.The protein of claim 1 in a pharmaceutically acceptable carrier.
 4. Theprotein of claim 2 in a pharmaceutically acceptable carrier.
 5. Theprotein of claim 1 immobilized on a solid phase.
 6. The protein of claim2 immobilized on a solid phase.
 7. A diagnostic kit comprising theprotein according to claim 1 and antibodies binding to said protein. 8.A diagnostic kit comprising the protein according to claim 2 andantibodies binding to said protein.